Dyspedic Mouse Skeletal Muscle Expresses Major Elements of the Triadic Junction but Lacks Detectable Ryanodine Receptor Protein and Function*

(Received for publication, July 23, 1996, and in revised form, November 18, 1996)

Edmond D. Buck Dagger , Hanh T. Nguyen §, Isaac N. Pessah Dagger par and Paul D. Allen §**

From the Dagger  Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, California 95616, the § Department of Cardiology, Children's Hospital, Boston, Massachusetts 02115, and ** Department of Anesthesia, Brigham and Women's Hospital, Boston, Massachusetts 02115

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
Acknowledgment
REFERENCES


ABSTRACT

The ry153 dyspedic mouse contains two disrupted alleles for ryanodine receptor type 1 (skeletal isoform of ryanodine receptor; Ry1R) resulting in perinatal death. In the present study, whole skeletal muscle homogenates and sucrose gradient-purified junctional sarcoplasmic reticulum from neonatal wild-type and dyspedic mice were assayed for biochemical and functional markers. Equilibrium binding experiments performed with 1-120 nM [3H]ryanodine reveal saturable high and low affinity binding to membrane preparations from wild-type mice, but not to preparations from dyspedic mice. Binding experiments performed with [3H]PN200 show a 2-fold reduction in [3H]PN200 binding capacity in dyspedic muscle, compared to age-matched wild-type muscle, with no change in receptor affinity. The presence or absence of proteins known to be critical for normal ryanodine receptor/Ca2+ channel complex function was assessed by Western blot analysis. Results indicate that FKBP-12, DHPRalpha 1, triadin, calsequestrin, SERCA1 (sarco(endo)plasmic reticulum Ca2+ ATPase), and skeletal muscle myosin heavy chain are present in both dyspedic and wild-type muscle. Only wild-type membranes showed immunoreactivity toward Ry1R antibody. Neither dyspedic nor wild-type mouse muscle showed detectable immunoreactivity toward Ry2R or Ry3R antibodies, even after sucrose gradient purification of sarcoplasmic reticulum. These results indicate that proteins critical for ryanodine receptor function are expressed in dyspedic skeletal muscle in the absence of Ry1R. Ca2+ transport measurements show that membranes from wild-type controls, but not dyspedic mice, release Ca2+ upon exposure to ryanodine. Dyspedic mice and cells derived from them serve as excellent homologous expression systems in which to study how Ry1R structure relates to function.


INTRODUCTION

The dyspedic mouse contains two disrupted alleles (ry153/ry153) for ryanodine receptor type 1 (skeletal isoform of ryanodine receptor; Ry1R)1 resulting in a birth lethal defect. Skeletal muscle from dyspedic mice lack excitation-contraction (E-C) coupling (1)2 but maintains many ultrastructural details of the triadic junction. Two important differences have been observed with thin section transmission and freeze fracture scanning microscopy of dyspedic muscle. First, dyspedic muscle lacks the regularly spaced array of junctional feet which span the gap between the t-tubule and SR membranes, significantly reducing the gap size (3).2 Second, dyspedic fibers lack the tetradic arrangement of dihydropyridine receptors which is characteristic of normal fibers (3). It is not yet known if dyspedic muscle lacking Ry1R expression results in altered expression of other key triadic proteins involved in modulating SR Ca2+ transport.

In addition to Ry1R, separate genes encode two other ryanodine receptors, namely the cardiac (Ry2R) and "brain" (Ry3R) isoforms (4-8). Ry1R is predominantly expressed in skeletal muscle and cerebellar Purkinje cells (9, 10). Ry2R is predominately expressed in cardiac tissue, where it functions as a Ca2+-induced Ca2+ release channel, but is also widely expressed in brain tissues (7, 9). In comparison, Ry3R is widely expressed at low levels in many non-muscle cells, in mammalian brain, and in very low levels in heart and skeletal muscle (11-13). However, its functional significance is presently unclear.

The development of Ry1R-null (dyspedic) mice (1)2 represents a significant advance in the goal to understand the molecular mechanisms regulating the function of Ry1R. Recent studies have shown that dyspedic mice die perinatally and lack skeletal E-C coupling (1).2 Dyspedic skeletal muscle fibers have also been found to have 30-fold less L-type Ca2+ entry current than control fibers, despite the presence of comparable levels of immobilization-resistant charge movement. Significantly, both voltage-dependent Ca2+ entry current and SR Ca2+ release are concomitantly restored in cultured dyspedic myotubes by micronuclear injection of Ry1R cDNA (14). Despite the loss of E-C coupling, dyspedic muscle fibers in culture exhibit small Ca2+ fluxes in response to caffeine and adenine nucleotide (14, 15). The pharmacological responses in cultured dyspedic muscle myotubes have been attributed to enhanced expression of Ry3R mRNA (15). However, there has been no direct evidence of Ry3R protein expression in dyspedic muscle.

The present study demonstrates for the first time that dyspedic muscle 1) exhibits a 2-fold reduction in [3H]PN200 binding capacity with a concomitant change in immunoreactive DHPRalpha 1 subunit expression, 2) lacks detectable levels of specific high or low affinity [3H]ryanodine-binding sites, 3) lacks immunoreactivity toward Ry1R, Ry2R, or Ry3R antibody, and 4) lacks a ryanodine-sensitive Ca2+ efflux pathway across SR membranes, compared with skeletal muscle from wild-type littermates. However, the complement of other triadic proteins known to be critical for normal E-C coupling (triadin, FKBP-12 (12-kDa FK506-binding protein), calsequestrin, SERCA1), and skeletal myosin heavy chain are shown to be expressed in dyspedic muscle.


EXPERIMENTAL PROCEDURES

Dyspedic Mice

Dyspedic mice were obtained as described elsewhere.2 Briefly, a 9-kilobase EcoRI-Tth111I genomic fragment which contains part of the skeletal Ry1 gene was screened from a 129/Sv mouse genomic library and was used to construct a targeting vector. A neomycin-resistance gene (Neo) was used to disrupt transcription of the mRNA and select for homologous recombinants. A herpes simplex virus thymidine kinase gene was used to reduce the number of non-homologously targeted clones. Both were under the control of a phosphoglycerate kinase promoter and were inserted at the KpnI and EcoRI sites, respectively. Approximately 2.5 × 107 J1-ES cells were electroporated with the targeting vector and subjected to double selection with G418 and FIAU. Homologously targeted ES clones were injected into blastocysts from C57BL/6 mice and transferred to pseudopregnant females. Chimeric males were bred to C57BL/6 females, and germline transmission was verified. Mutant mice homozygous for the Ry1 targeted allele were obtained from heterozygous matings and verified by PCR and Southern blot.

Tissue Preparation

Crude and sucrose gradient-purified membrane fractions were prepared from wild-type (ry1+/+) and dyspedic (ry1-/-) neonates under identical conditions. Immediately after birth, wild-type animals were euthanized by cervical dislocation. Tail tissue from each sample (dyspedic and wild-type) was removed for PCR analysis. The legs were removed from each animal at the hip or shoulder, trimmed of feet, skinned, and frozen in liquid nitrogen. Wild-type or dyspedic tissues were then pooled and prepared in the following manner. Legs were thoroughly homogenized on ice using a Polytron at high speed in 30-s bursts in homogenization buffer consisting of 300 mM sucrose, 25 mM Hepes, pH 7.1, 200 µM phenylmethylsulfonyl fluoride (prepared immediately before use), and 10 µg/ml leupeptin. Bone fragments were removed by gentle centrifugation at 50 × g for 2 min at 4 °C. The homogenate was centrifuged at 110,000 × g for 90 min at 4 °C. The resulting pellet was resuspended in 10% sucrose, 25 mM Hepes, pH 7.1, to a protein concentration of approximately 3 mg/ml, snap-frozen in liquid nitrogen, and stored at -80 °C until used.

In some experiments, enrichment of the heavy SR membrane fraction from normal and dyspedic homogenates was achieved by sucrose gradient sedimentation. Crude membrane homogenates (3-5 ml) were layered onto a discontinuous sucrose gradient (2 ml 27%, 3 ml 32%, 3 ml 34%, 3 ml 38%, and 2 ml 45%) and centrifuged at 70,000 × g for 16 h at 4 °C. Membrane fractions removed from the 38-45% sucrose step interface were pooled, diluted to 10% sucrose with a 25 mM Hepes pH 7.1 buffer, homogenized, and pelleted at 110,000 × g for 90 min at 4 °C. The pellets were resuspended in 10% sucrose, 25 mM Hepes, pH 7.1, to a protein concentration of approximately 1 mg/ml, snap-frozen in liquid nitrogen, and stored at -80 °C until used.

The following tissue preparations were used as reference standards for Western blot analysis. Rabbit fast skeletal muscle junctional SR was isolated according to Saito et al. (16), rat cardiac SR membranes were isolated according to Feher et al. (17), and avian SR was isolated from pectoralis muscles according to Airey et al. (18). A whole membrane homogenate was prepared from rat testicles following the procedure described for wild-type mouse muscle, with the exception that the 50 × g centrifugation step was replaced by a 30-min, 8000 × g centrifugation step. Protein was quantitated in all preparations by the method of Lowry (19) using bovine serum albumin as a standard.

Radioligand Binding Assay

High and low affinity binding of [3H]ryanodine (84 Ci/mmol, DuPont NEN) to 0.2 mg/ml skeletal muscle microsomes was measured in the presence of 250 mM KCl, 15 mM NaCl, 20 mM Hepes, pH 7.1, 50 µM Ca2+, 1 nM [3H]ryanodine, and 0.5-120 nM unlabeled ryanodine (Calbiochem). The reaction was initiated by the addition of tissue and allowed to equilibrate at 37 °C for 3 h. Nonspecific binding was assessed in the presence 260 nM unlabeled ryanodine. Separation of bound and free ligand was performed by filtration through Whatman GF/B glass fiber filters using a Brandel (Gaithersburg, MD) cell harvester. Filters were washed with three volumes of 0.5 ml of ice-cold wash buffer containing 20 mM Tris-HCl, 250 mM KCl, 15 mM NaCl, 50 µM CaCl2, pH 7.1, and placed into vials containing 5 ml of scintillation mixture (Ready Safe, Beckman). Binding of radioligand to muscle membranes was determined by scintillation spectrometry. Kd and Bmax values were derived from Scatchard analysis of the binding data.

Specific binding of 0.06-5.0 nM [3H]PN200 (83 Ci/mmol, DuPont NEN) to the alpha 1 subunit of the L-type Ca2+ channel (the dihydropyridine receptor) was measured in the presence of 140 mM NaCl, 15 mM KCl, 20 mM Hepes, pH 7.0, and 0.2 mg/ml skeletal muscle homogenate. The reaction was initiated by the addition of tissue and allowed to equilibrate at 22 °C for 30 min in the dark. Paired nonspecific controls were measured in the presence of 10 µM nifedipine. Separation of bound and free ligand was performed as described for high affinity [3H]ryanodine binding except that the filters were washed with 5 × 2 ml of wash buffer. Kd and Bmax values were derived from Scatchard analysis of the binding data.

Electrophoresis and Immunoblot Analysis

Constituent proteins from membrane preparations were resolved on 3-10% gradient, 4-20% gradient, or 7% isocratic gels by the method of Laemmli (20). Gels were either stained with silver (Silver Stain Plus, Bio-Rad) or Stains All (Sigma), or transferred onto polyvinylidene difluoride (PVDF) membranes (Immobilon-P, Millipore) for subsequent Western blot analysis. For Western blots, proteins were electroblotted (Mini Trans-Blot, Bio-Rad) overnight at 30 V, followed by a 60-min fast transfer at 100 V. Nonspecific antibody binding was blocked by incubating blots for 1 h at 37 °C in TTBS buffer solution (20 mM Tris-HCl, 500 mM NaCl, 0.5% Tween 20, pH 7.5) with the addition of 5% bovine serum albumin or 5% nonfat dry milk. Specific binding of the primary antibody of interest was performed by incubating the blots for 1 h at 37 °C in TTBS buffer in the presence of 1% bovine serum albumin and antibody. Resulting immunoblots were labeled with horseradish peroxidase-conjugated goat anti-mouse (Sigma) or donkey anti-rabbit (Amersham) secondary antibody for 1 h at 37 °C and then visualized using either colorimetric (TMB, Vector Laboratories) or chemiluminescent (ECL, Amersham) techniques. In some cases, exposed films from ECL were quantitated using a densitometer (model PC9310, Shimadzu). Nonspecific binding of secondary antibodies to membrane preparations was minimized by performing a dilution series in the absence of primary antibody. Antibodies were purchased or generously provided as follows: Ry1 (34C), generous gift of Dr. J. Sutko; Ry2 (C3-33), generous gift of Dr. G. Meissner; Ry3, generous gift of Dr. V. Sorrentino; DHPRalpha 1 (MA3-920), Affinity BioReagents; triadin (11G12A8), generous gift of Dr. K. Campbell; FKBP-12, generous gift of Dr. M. Harding, Vertex Pharmaceuticals; SERCA1 (MA3-911), Affinity BioReagents; myosin heavy chain, Affinity BioReagents (1130-P; Biocytex Biochemicals, San Diego, CA).

Transport Measurements

Ca2+ flux measurements using microsomal membranes from wild-type or dyspedic mouse skeletal muscle were performed fluorometrically (SLM AB-2, SLM-Aminco). Briefly, 50 µg of skeletal muscle microsomal membranes were equilibrated to 37 °C in a buffer consisting of 92 mM KCl, 7.5 mM Na4P2O7, 20 mM MOPS, pH 7.0, 0.01% NaN3, 2-10 µM Ca2+, and 0.5 µM fluo-3. A coupled enzyme system (20 µg/ml creatine phosphokinase and 5 mM phosphocreatine) was present to maintain ATP concentrations. Loading of Ca2+ was initiated by the addition of 1 mM MgATP. Transport of Ca2+ into or out of microsomal membranes was determined by following changes in the fluo-3 fluorescence intensity (excitation at 500 nm, emission at 530 nm). The presence of Na4P2O7 in the transport buffer maintained a linear dye response at added Ca2+ concentrations up to 8 µM. Ca2+ efflux was initiated by the addition of 20 or 200 µM ryanodine. Ca2+ accumulation by microsomal stores was verified by addition of 2 µg/ml 4-bromo-A23187. Linearity of fluo-3 emission with increasing Ca2+ concentration was verified after each experiment by adding known aliquots from a National Bureau of Standards Ca2+ stock.


RESULTS AND DISCUSSION

Silver Stain of Dyspedic and Wild-type Control Mouse Skeletal Muscle

Wild-type and dyspedic neonatal mouse skeletal muscle proteins, resolved on 3-10% Laemmli gels and visualized by silver stain, are shown in Fig. 1. Lanes containing either dyspedic (dys) or wild-type (w-t) preparations exhibit a similar pattern of staining and density of protein bands, with the major exception of a band in wild-type lanes corresponding in size to the Ry1R protomer found in junctional rabbit skeletal muscle SR (jsr lane, marked with arrow).


Fig. 1. SDS-PAGE of wild-type and dyspedic skeletal muscle proteins reveals the presence and absence of Ry1R, respectively, by silver stain. SDS-PAGE was performed with wild-type and dyspedic mouse skeletal muscle preparations according to the method of Laemmli using 3-10% gradient gels. Proteins were visualized using the Silver Stain Plus kit (Bio-Rad). jsr, 4 µg of rabbit skeletal junctional SR; dys, 4, 8, and 12 µg of dyspedic mouse microsomal membranes, respectively; w-t, 4, 8, and 12 µg of wild-type mouse microsomal membranes, respectively; std, protein standards. Molecular mass markers (indicated by arrows along left side of figure) are 170 kDa (reduced alpha 2-macroglobulin), 116 kDa (beta -galactosidase), 85 kDa (fructose 6-phosphate), 55 kDa (glutamate dehydrogenase), 39 kDa (aldolase), and 26 kDa (triosephosphate isomerase).
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The Ry1R, Ry2R, and Ry3R Isoforms Are Not Detectable in Dyspedic Mouse Skeletal Muscle

The equilibrium binding of [3H]ryanodine to neonatal wild-type and dyspedic skeletal muscle microsomes was investigated under conditions that stabilize high affinity binding of the alkaloid to ryanodine receptors (21). Fig. 2 shows that membranes prepared from dyspedic skeletal muscle do not possess specific high affinity [3H]ryanodine-binding sites, whereas preparations of newborn wild-type muscle exhibit 148 ± 16 fmol/mg of high affinity [3H]ryanodine binding with a Kd of 6.9 ± 1.3 nM (mean Bmax from three independent preparations, each performed in duplicate). Scatchard analysis of data from control muscle membranes is best fit using a two-site model, with a second site having lower affinity (Kd = 20.3 nM) and Bmax of 305 ± 94 fmol/mg of protein. Experiments designed to measure the presence of binding sites having lower affinity were assessed using [3H]ryanodine concentrations from 250-1600 nM (specific activity 1.2 Ci/mmol) and revealed that while wild-type preparations were fully saturated, dyspedic preparations failed to show any specific binding (data not shown).


Fig. 2. Dyspedic skeletal muscle membranes lack detectable high affinity binding sites for [3H]ryanodine. Specific binding of [3H]ryanodine to membrane preparations from wild-type and dyspedic mice were performed as described under "Experimental Procedures" in the presence of 1 nM [3H]ryanodine and 0.5-120 nM unlabeled ryanodine. Nonspecific binding was determined in the presence of 260 nM unlabeled ryanodine. Scatchard analysis (inset) of specific binding found in wild-type preparations was best fit by a two-site model. The high affinity site gave a Bmax of 148 ± 16 fmol/mg of protein and a Kd of 6.9 ± 1.3 nM; the lower affinity site gave a Bmax of 305 ± 94 fmol/mg of protein and a Kd of 21 ± 1.8 nM (pooled data from three independent experiments performed in duplicate). In contrast, specific ryanodine binding was not detected with dyspedic mouse skeletal muscle (three independent preparations).
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The binding data presented above suggest that dyspedic muscle either lacks general expression of ryanodine receptor isoforms or, if these proteins (e.g. Ry3R) are expressed, they fail to measurably recognize the alkaloid. We directly examined whether or not dyspedic muscle expresses any of the known ryanodine receptor isoforms by Western blot analysis. Proteins from wild-type and dyspedic skeletal muscles were resolved by SDS-PAGE and transferred overnight onto PVDF membranes as described under "Experimental Procedures." Blots were probed using antibodies selective for either Ry1, Ry2, or Ry3 receptors. Specific antibody labeling was visualized using a highly sensitive chemiluminescent (ECL) technique.

Blots probed with a Ry1R-selective mouse monoclonal antibody (22) stain positive for the ~560-kDa protomer in a protein concentration-dependent manner with preparations from wild-type neonatal mice and with rabbit junctional SR (Fig. 3, top panel, w-t and jsr lanes). In comparison, dyspedic skeletal muscle preparations lack any detectable immunoreactivity with the Ry1R antibody (Fig. 3, top, dys lanes), as does rat cardiac SR (Fig. 3, top, crd lane).


Fig. 3. Dyspedic muscle membranes do not express detectable levels of Ry1R, Ry2R, or Ry3R proteins by Western blot analysis. Constituent proteins from dyspedic and wild-type muscle membranes were resolved by SDS-PAGE using 3-10% gradient gels and transferred overnight onto PVDF membranes. Secondary antibody was visualized using enhanced chemiluminescent (ECL, Amersham) methods. Arrow along left side of blot indicates position of protein band of interest. Top panel, Ry1R Western blot. jsr, 5 µg of rabbit skeletal junctional SR; crd, 5 µg of rat cardiac SR; dys (left to right), 5, 10, and 15 µg of dyspedic muscle protein; w-t (left to right), 5, 10, and 15 µg of wild-type muscle protein. Middle panel, Ry2R Western blot. crd, 5 µg of rat cardiac SR; dys (left to right), 5, 10, and 15 µg of dyspedic muscle protein; ctl (left to right), 5, 10, and 15 µg of wild-type muscle protein. Bottom panel, Ry3R Western blot. jsr, 10 µg of rabbit skeletal muscle protein; avi, 15 µg of avian pectoralis SR; dys, 15 µg of dyspedic muscle protein; w-t, 15 µg of wild-type protein; tst, 20 µg of rat testicular protein.
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As expected, Western blot analysis using a Ry2R-selective monoclonal antibody (23) shows an absence of reactivity with both dyspedic and wild-type skeletal muscle preparations (Fig. 3, middle panel, dys and w-t lanes). However, the antibody strongly recognizes the Ry2R protein found in rat cardiac preparations, which is included as a positive control (Fig. 3, middle, crd lane).

Western blot analysis using a Ry3R-selective polyclonal antibody (8) does not recognize any protein associated with either the dyspedic or wild-type membrane preparations (Fig. 3, bottom panel, dys and w-t lanes), nor does it recognize any proteins associated with rabbit fast skeletal muscle junctional SR (Fig. 3, bottom, jsr lane). However, the antibody strongly recognizes the Rybeta receptor isoform found in avian pectoralis muscle, which possesses >80% sequence homology with the mammalian Ry3R "brain" isoform (Fig. 3, bottom, avi lanes). The antibody also recognizes Ry3R protein in rat testicular tissue (Fig. 3, bottom, tst lane), which has been shown to express very high levels of Ry3R mRNA (8). Sequential labeling experiments with Ry1R and Ry3R antibodies reveal that the Ry3R protomer found in avian muscle and rat testicular tissue migrates with a smaller apparent size than Ry1R.

Expression of Ry1R and Ry3R proteins was further investigated using SR membrane fractions purified by sucrose density gradient centrifugation. Gradient fractions isolated at the 38-45% sucrose interface and within the 45% sucrose layer were analyzed by Western blot using Ry1R- and Ry3R-selective antibodies. Fig. 4top panel, shows that lanes containing either whole membrane fractions (memb), 38-45% sucrose gradient fractions (38-45%), or the 45% sucrose fraction (45%) react with the Ry1R-selective antibody only in preparations obtained from wild-type muscle, whereas dyspedic muscle preparations completely lack immunoreactivity toward this antibody. In comparison, no immunoreactive protein could be detected with the Ry3R polyclonal antibody in the purified fractions from either the wild-type or dyspedic muscle preparations (Fig. 4, bottom panel). Multiple bands labeled by the Ry1R-selective antibody in the 38-45% sucrose gradient lane of wild-type muscle (Fig. 4, top) are most likely proteolytic fragments of the ryanodine receptor with Mr estimated to be 250,000 and 300,000, which is reflective of the fragmentation pattern of Ry1R induced by trypsin (24) or calpain (25) digestion. The sedimentation density of the vesicles could be related to the degree of proteolysis of Ry1R, especially since Ry1R fragmentation was not observed within the 45% sucrose gradient fraction.


Fig. 4. Sucrose density gradient purification does not reveal ryanodine receptor proteins by Western blot analysis in dyspedic membranes. Dyspedic and wild-type mouse skeletal membranes were further purified by sucrose density gradient centrifugation as described under "Experimental Procedures." Purified membranes were transferred onto PVDF membranes and Western blot analysis performed as in Fig. 3. Arrow on left side of blot indicates position of protein band of interest. Top panel, Ry1R Western blot. Bottom panel, Ry3R Western blot. Lane markers are as follows: jsr, 1 µg of rabbit skeletal SR; avi, 15 µg of avian pectoralis SR; memb, 15 µg of whole muscle homogenate from either dyspedic (dys) or wild-type (w-t) muscle; 38-45%, 15 µg of purified membranes from corresponding to pooled sucrose gradient fractions from either dyspedic or wild-type muscle; 45%, 15 µg of particulate fraction from bottom of 45% sucrose layer from either dyspedic or wild-type muscle. The multiple bands labeled in the 38-45% w-t lane are proteolytic fragments generated during the purification process.
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The results presented here and elsewhere (1, 3, 15)2 clearly demonstrate that dyspedic mouse skeletal muscle does not express the skeletal isoform (Ry1R) of the ryanodine receptor. In the present study, specific antibodies fail to detect the presence of Ry1R, Ry2R or, more importantly, Ry3R protein in the neonatal muscle, even when the highly sensitive ECL technique is used in conjunction with sucrose gradient purification of SR. Furthermore, analysis of high and low affinity [3H]ryanodine binding fails to support the presence of any known ryanodine receptor proteins in these preparations. Since dyspedic and wild-type skeletal muscle preparations are from newborn mice, SR volume (26) and frequency of SR/t-tubule junctions are much less than those found in adult mouse muscle (26-28). This morphological difference, along with results from the Western blots presented here, suggests that neonatal mouse skeletal muscle either does not express Ry3R protein or expresses it at levels below detection limits.

The question of whether dyspedic muscle expresses an alternate ryanodine receptor isoform (Ry2R or Ry3R) is important, since responses to Ca2+, caffeine, and ryanodine have been observed in cultured dyspedic myotubes (1, 14, 15). However, the total Ca2+ released in response to these ligands was observed to be 10-15-fold less than that seen in normal myotubes, and the time course for release was significantly slower (1, 14). Furthermore, cultured dyspedic myotubes have been reported to lack responsiveness to a second addition of caffeine when ryanodine is introduced between caffeine additions (14, 15). Analysis of RNA from cultured myotubes using reverse transcription-polymerase chain reaction have revealed increased Ry3R mRNA expression in cultured dyspedic myotubes (15). Based on these lines of evidence, the Ca2+ fluxes seen in dyspedic muscle cells have been attributed to up-regulation of Ry3R protein as a consequence of Ry1R deletion (15). In support of this hypothesis, Conti et al. (12) recently described a differential distribution of the Ry3R gene product in various adult mammalian skeletal muscles using Western blot analysis and in situ hybridization. In that study, bovine hind limb, Type II rat skeletal muscle, and diaphragm from the mouse, rabbit, and cow were all found to differentially express Ry3R protein. Their results indicate that expression of Ry3R protein differs dramatically among muscle types within a single species and suggest that these differences may also be species-specific. Additionally, Ry3R protein expression was found to be concentrated to the terminal cisternae of SR in bovine diaphragm muscle. However, the significance of Ry3R expression in skeletal muscle remains unclear. In the Ry3-null mouse, the protein does not appear to be essential for E-C coupling or normal muscle development (12), but the maximum Ca2+-induced Ca2+ release response obtained at high Ca2+ in permeabilized muscle fibers is reduced by ~25% (12).

DHPR Protein Expression Is Diminished in Dyspedic Mouse Skeletal Muscle

The functional importance of DHP receptors in E-C coupling has been amply demonstrated in studies of the dysgenic mouse, which has been shown to lack expression of DHPRalpha 1 subunit protein and DHPR function (29). Like dysgenic muscle (30), dyspedic muscle lack tetrads found in normal skeletal muscle. When cultured dysgenic myotubes are transfected with cDNA coding for the DHPRalpha 1 subunit, E-C coupling is restored and the DHP receptors align as tetrads revealing the same structures as seen in wild-type muscle (31). Electron microscopic studies of dyspedic and dysgenic skeletal muscle has revealed that neither Ry1R nor DHPRalpha 1 expression is the signal required for close apposition of the t-tubule and SR membranes and the subsequent co-localization of the remaining triadic cytoskeletal elements (3). Interestingly, while disruption of Ry1R gene expression does not preclude the close proximity of the triadic membranes, it does prevent DHPR tetrad formation (3).

Recently, Nakai et al. (14) reported that whole cell L-type Ca2+ currents in cultured dyspedic myotubes are reduced ~30-fold when compared to wild-type controls. Micronuclear injection of Ry1R cDNA into dyspedic myotubes reconstituted L-type inward Ca2+ current to ~40% of control myotubes and restored E-C coupling with no concomitant change in immobilization-resistant charge movement (Qmax). Their results indicate that E-C coupling is restored in dyspedic myotubes upon expression of Ry1R, and that Ry1R expression does not significantly alter the level of expression and/or targeting of DHPR alpha 1 subunit protein to the surface membrane, but rather is important in conveying reciprocal regulation for DHPR Ca2+ channel function. However, since Qmax is normalized to membrane surface area, the absolute change in the amount of DHPR expression in dyspedic muscle remains unclear.

To more directly ascertain levels of DHPR expression in dyspedic skeletal muscle, the specific binding of [3H]PN200 (0.06-5.0 nM) is compared in whole membrane preparations from dyspedic and wild-type skeletal muscle. Fig. 5A shows that compared to those from wild-type muscle, the density of specific [3H]PN200-binding sites is reduced ~50% in dyspedic muscle preparations without a significant change in Kd (Fig. 5B). Three independent experiments using preparations from different animals reveal that the density of [3H]PN200-binding sites is consistently reduced in the dyspedic preparations compared to wild-type (168 ± 7 fmol/mg of protein and 331 ± 29 fmol/mg of protein, respectively). Western blot analysis with a DHPRalpha 1-selective antibody (32) reveals that while both dyspedic and wild-type muscle preparations exhibit immunoreactivity at ~170 kDa, dyspedic preparations stain to a lesser degree compared to wild-type lanes containing equal amounts of whole membrane-bound protein (Fig. 6, DHPRalpha 1 blot). Consistent with the [3H]PN200 binding data shown in Fig. 5, densitometric analysis of ECL radiograms reveals that the immunoreactive band at ~170 kDa corresponding to DHPRalpha 1 is approximately 50% less dense in the dyspedic preparations, regardless of the amount of protein loaded on the gel (data not shown).


Fig. 5. Dyspedic mouse membranes exhibit reduced capacity to bind [3H]PN200. A, binding of [3H]PN200 to dyspedic and wild-type mouse membrane preparations was performed in the presence of 0.06-5.0 nM [3H]PN200. Nonspecific binding was determined under identical conditions with the addition of 10 µM nifedipine. B, Scatchard analysis of binding curves to wild-type membranes reveals a Bmax of 331 ± 29 fmol/mg of protein and a Kd of 0.44 ± 0.12 nM, whereas dyspedic mouse muscle reveals a Bmax of 168 ± 7 fmol/mg of protein and a Kd of 0.40 ± 0.06 nM. Data presented are representative of n = 3 independent measurements, each performed in duplicate.
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Fig. 6. Dyspedic skeletal muscle expresses key triadic proteins. Constituent proteins from wild-type and dyspedic mouse membranes were resolved by Laemmli SDS-PAGE using 3-10% gradient, 4-20% gradient, or 7% isocratic gels and transferred overnight onto PVDF membranes. Secondary antibody was visualized using either chemiluminescent (ECL, Amersham) or colorimetric (TMB, Vector Laboratories) methods. Arrow on left side of each blot indicates position of protein band of interest. DHPRalpha 1 Western blot, jsr, 10 µg of rabbit skeletal SR; std, protein standard from overexposed blot showing position of 170-kDa marker; dys, 5, 10, and 15 µg of dyspedic membranes; w-t, 5, 10, and 15 µg of wild-type membranes. FKBP-12 Western blot, jsr, 5 µg of rabbit skeletal junctional SR; dyspedic, 15 µg of dyspedic membranes; w-t, 15 µg of wild-type membranes. Triadin Western blot, jsr, 1 µg of rabbit skeletal junctional SR; dys, 10 and 15 µg of dyspedic membranes; w-t, 10 and 15 µg of wild-type membranes. Calsequestrin blot (visualized with Stains All), dys, 10 µg of dyspedic membranes; w-t, 10 µg of wild-type membranes; jsr, 1 µg of rabbit skeletal junctional SR. SERCA1 Western blot, jsr, 1 µg of rabbit skeletal junctional SR; dys, 10 µg of dyspedic membranes; w-t, 10 µg of wild-type membranes. Myosin Western blot, jsr, 5 µg of rabbit skeletal junctional SR; w-t, 10 µg of wild-type membranes; dys, 10 µg of dyspedic membranes. Blot is visualized colorimetrically (TMB, Vector Laboratories).
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The observation of reduced DHPRalpha 1 expression in dyspedic skeletal muscle compared to age-matched wild-type muscle could reflect either 1) a lower density of DHP receptors within the t-tubule membrane, or 2) a decrease in t-tubule surface area, both of which would lower the total DHPR capacity of dyspedic membranes. It is unlikely that DHP receptor density is reduced in t-tubule membranes, since Qmax is normalized to membrane surface area and Nakai et al. reported no significant difference in Qmax between dyspedic and wild-type myotubes (14). It is more likely that the lower density of [3H]PN200-binding sites can be accounted for by a decrease in t-tubule membrane surface area, since t-tubules are significantly less extensive in dyspedic muscle compared to wild-type muscle.3

In the report by Nakai et al. (14), L-type Ca2+ currents in cultured dyspedic myotubes were restored to ~40% of those seen in cultured wild-type myotubes by microinjection of Ry1 cDNA. This increase could be attributed to 1) an increase in t-tubule membrane surface area (since DHP receptor density is unaltered by Ry1 expression), or 2) to the establishment of a requisite communication between Ry1R and DHPR. The present results show that DHPRalpha 1 protein levels are reduced ~2-fold in dyspedic muscle compared to wild-type muscle. This difference cannot account for the dramatic 30-fold reduction in L-type Ca2+ current seen between wild-type and dyspedic myotubes, and as stated above, is more likely the result of a less developed t-system. This result indicates that deletion of Ry1R protein expression eliminates some form of reciprocal (retrograde) regulation on the DHPR complex, and therefore, it is unlikely that the increase in L-type Ca2+ current seen in cultured dyspedic myotubes in which Ry1 expression has been restored is due to an increase in membrane surface area. This interpretation is supported by the observations that 1) the L-type Ca2+ current in dyspedic myotubes prior to microinjection is much less than 50% of wild-type myotubes, and 2) under ideal conditions (100% expression of Ry1R in dyspedic myotubes) the L-type Ca2+ current would not be expected to return to 100% of that seen in wild-type myotubes, since dyspedic myotubes contain significantly less t-tubule membrane.

Triadic Proteins Critical for E-C Coupling Are Expressed in Dyspedic Mouse Skeletal Muscle

Whether or not Ry1R deletion alters the expression of other key triadic proteins normally found in skeletal muscle has not been addressed. In addition to DHPRalpha 1, several proteins localized at the muscle triad have been shown to directly or indirectly modulate the function of Ry1R. Therefore, the presence of FKBP-12, triadin, calsequestrin, SERCA1, and myosin heavy chain in dyspedic mouse skeletal muscle membranes was examined by Western blot analysis.

A high affinity interaction between Ry1R and FKBP-12 (12-kDa FK506-binding protein) has been shown to be essential for stabilizing the full conductance gating behavior of the SR channel (33-35). Blots stained with a monoclonal antibody directed against FKBP-12 reveal the presence of this protein (Mr ~14,000) in both dyspedic and wild-type muscle membranes (Fig. 6, FKBP-12 blot, dys and w-t lanes). Interestingly, the amount of FKBP-12 associated with dyspedic membrane preparations was consistently observed to be greater than the corresponding wild-type preparations. The underlying reason for this increase in FKBP-12 in dyspedic membranes is unknown. Whether FKBP-12 is elevated in dyspedic muscle membranes as a direct result of loss of muscle function or is associated with infiltration of immunocompetent cells or erythrocytes (36) remains to be determined. However, the latter possibility is unlikely since microscopic examination of muscle does not reveal evidence of an inflammatory response or increased numbers of red cells in dyspedic muscle. In agreement with previous reports (33, 37), purified rabbit junctional SR preparations contain FKBP-12 (Fig. 6, FKBP-12 blot, jsr lane).

Triadin, a 95-kDa protein initially identified by Caswell et al. (38) and cloned by Campbell and co-workers (39), appears to form an association with Ry1R and calsequestrin (40-42). Blots probed with a monoclonal antibody directed toward triadin (39) and visualized by ECL reveal the presence of this protein in both dyspedic and wild-type skeletal muscle (Fig. 6, triadin blot, dys and w-t lanes, respectively), as well as in rabbit junctional SR (Fig. 6, triadin blot, jsr lane). The low density of labeling seen in the dys and w-t lanes, compared to the junctional SR lane, reflects the lower density of this protein found in the whole muscle preparation.

In addition to its role in enhancing the Ca2+ loading capacity of SR, calsequestrin appears to elicit a signal that is communicated to Ry1R during Ca2+ release (43). The presence of calsequestrin in dyspedic and wild-type skeletal muscle was probed using Stains All as described by Campbell et al. (44). Proteins were resolved on 3-10% Laemmli gels and stained for 48 h with Stains All, followed by de-staining for 1-2 h to resolve an intensely blue band at Mr ~60,000. The calsequestrin gel shown in Fig. 6 reveals the presence of a blue band corresponding to the location of calsequestrin in lanes containing either rabbit junctional SR (jsr), dyspedic (dys), or wild-type (w-t) muscle proteins. Note that the lane containing rabbit junctional SR stains a comparatively broad band, reflecting the higher density of this protein found in the purified preparation.

The presence of two additional proteins critical for normal muscle function was probed by Western blot analysis. The SERCA1 and myosin blots in Fig. 6 show that antibodies selective for SERCA1 (2) and myosin heavy chain recognize their respective targets in both wild-type and dyspedic membrane preparations. Since the relative density of protein labeling by these antibodies reflects differences in protein content within these preparations, SERCA1 stains to a much higher degree in purified junctional SR preparations (SERCA1 blot, jsr lane) than in the whole muscle preparations (SERCA1 blot, dys and w-t lanes). In comparison, antibody specific for myosin heavy chain reveals a higher density of myosin in the dyspedic and wild-type preparations (myosin blot, dys and w-t lanes, respectively) since these are whole membrane preparations.

Results presented here using Western blot analysis reveal that FKBP-12, triadin, and calsequestrin are all expressed in dyspedic mouse skeletal muscle preparations. Interestingly, while FKBP-12 expression is increased and DHPR expression is decreased in dyspedic mouse skeletal muscle as compared to wild-type membranes, the pattern of expression of key triadic proteins remains and suggests that the molecular components required to form a functional triadic complex will also be present in myogenic cell lines produced using the Ry1R gene targeting approach.

Dyspedic Mouse Skeletal Muscle Microsomes Do Not Exhibit Ryanodine-induced Ca2+ Release

[3H]Ryanodine binding and Western blot analysis reveal a lack of ryanodine receptor expression in dyspedic muscle. As a functional correlate, Ca2+ flux measurements were performed and ryanodine-induced Ca2+ release was assayed using microsomal membranes from dyspedic and wild-type muscle. Ca2+ transport across isolated microsomal membranes was assessed fluorometrically with the dye fluo-3. In the presence of 7.5 mM sodium pyrophosphate in the buffer, Ca2+ additions of 1-8 µM produced linear responses from the dye (Fig. 7A). Addition of MgATP in the presence of a regenerating system initiates active accumulation of Ca2+ into membrane vesicles (Fig. 7, B and C, insets). Under the experimental conditions used, wild-type and dyspedic membranes could be loaded with similar amounts of Ca2+ (100 and 74 nmol/mg of protein, respectively). Addition of 20 µM ryanodine to Ca2+-loaded microsomes from wild-type muscle results in release of approximately 40% of the intravesicular Ca2+, and this effect is fully blocked by pretreatment with 10 µM ruthenium red (Fig. 7B, traces a and b, respectively). Ryanodine-induced Ca2+ release from control microsomes can also be blocked by prior addition of 1 µM neomycin (data not shown). Addition of 200 µM ryanodine to wild-type vesicles elicited a biphasic effect on Ca2+ transport, initially inducing Ca2+ release followed by Ca2+ reaccumulation (Fig. 7B, trace c). This result is in agreement with results commonly obtained with rabbit junctional SR and reflects a sequential action of ryanodine at its binding sites. In contrast, membrane microsomes obtained from dyspedic muscle do not release Ca2+ in response to 200 µM ryanodine (Fig. 7C). The absence of a transient response to 200 µM ryanodine in the dyspedic preparation is further evidence that these muscle microsomes do not contain measurable ryanodine-sensitive Ca2+ effluxes.


Fig. 7. Ryanodine stimulates Ca2+ release from wild-type control, but not dyspedic mouse microsomal preparations. Ca2+ release was measured from actively loaded dyspedic mouse or wild-type mouse skeletal muscle microsomes using the fluorescent dye fluo-3 as described under "Experimental Procedures." After loading, Ca2+ release was initiated by the addition of either 20 or 200 µM ryanodine. A, Ca2+ aliquots (1 µM) were added to the complete transport buffer in the presence of 50 µg of wild-type membranes and 4-bromo-A23187 to verify the linearity of the fluo-3 dye response over the ranges used. While the dye approached saturation at Ca2+ concentrations greater than 10 µM, the response remained linear throughout the range of the assay. B, Ca2+ release induced by 20 µM ryanodine (trace a) from wild-type mouse microsomes is inhibited by 10 µM ruthenium red (trace b). Additionally, 200 µM ryanodine induces a transient Ca2+ release in wild-type mouse microsomes (trace c). C, 200 µM ryanodine does not induce Ca2+ release in dyspedic mouse microsomes. Insets show loading phase.
[View Larger Version of this Image (13K GIF file)]


The results presented above indicate that while dyspedic mouse skeletal muscle does not express Ry1R, it does express the balance of the major triadic elements critical for E-C coupling. These findings, along with the Ca2+ flux measurements presented above, indicate that the mouse model is an ideal system with which to examine Ca2+ regulation in skeletal muscle using a homologous expression system and transgenic approaches.


FOOTNOTES

*   This work was supported in part by grants from the Muscular Dystrophy Association and the Brigham and Women's Hospital Anesthesia Foundation (to P. D. A.), the American Heart Association, California Affiliate (to I. N. P. and E. D. B.), and United States Public Health Service Grants 1RO1-AR43140 (to P. D. A. and I. N. P.) and 1RO1-E505002 (to I. N. P.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
   Supported by the Boston Heart Foundation.
par    To whom correspondence should be addressed. Tel.: 916-752-6696; Fax: 916-752-4698.
1   The abbreviations used are: Ry1R, ryanodine receptor, skeletal isoform; Ry2R, ryanodine receptor, cardiac isoform, Ry3R, ryanodine receptor, brain isoform; DHP, dihydropyridine; DHPR, dihydropyridine receptor; E-C, excitation-contraction; ECL, enhanced chemiluminescence; PVDF, polyvinylidene difluoride; PCR, polymerase chain reaction; PAGE, polyacrylamide gel electrophoresis; SR, sarcoplasmic reticulum; t-tubule, transverse tubule; MOPS, 4-morpholinepropanesulfonic acid.
2   H. T. Nguyen, E. D. Buck, S. Mukherjee, S. Schieferl, P. C. Dolber, J. R. Sommer, I. N. Pessah, and P. D. Allen, submitted for publication.
3   C. Franzini-Armstrong, personal communication.

Acknowledgment

We thank Lili Chen for technical assistance.


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