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(The FASEB Journal. 2002;16:943-949.)
© 2002 FASEB

Characterization of aquaporin-4 in muscle and muscular dystrophy

RACHELLE H. CROSBIE1, SHERRI A. DOVICO, JASON D. FLANAGAN, JEFFREY S. CHAMBERLAIN*, CHARLOTTE L. OWNBY{dagger} and KEVIN P. CAMPBELL2

Howard Hughes Medical Institute, Department of Physiology and Biophysics, Department of Neurology, University of Iowa College of Medicine, Iowa City, Iowa, USA;
* Department of Neurology, K24b Health Sciences Center, University of Washington School of Medicine, Seattle, Washington, USA; and
{dagger} Department of Physiological Sciences, Oklahoma State University, Stillwater, Oklahoma, USA

2Correspondence: Howard Hughes Medical Institute, University of Iowa College of Medicine, 400 Eckstein Medical Research Building, Iowa City, IA 52242, USA. E-mail: kevin-campbell{at}uiowa.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Aquaporins are a growing family of transmembrane proteins that transport water and, in some cases, glycerol and urea across cellular membranes. Aquaporin-4 (AQP4) is enriched at the sarcolemma of skeletal muscle and may play a role in accommodating the rapid changes in cell volume and hydrostatic forces that occur during contraction in order to prevent damage to the sarcolemma. Recent evidence has shown that AQP4 is absent in dystrophin-deficient mdx mice, suggesting that AQP4 associates with dystrophin and has a role in the dystrophic process. To examine the relationship between aquaporins and muscle disease, and between aquaporins and dystrophin, we have investigated aquaporin expression in various mouse models of muscular dystrophy and cardiomyopathy before and after the onset of pathology. We find that AQP4 is expressed in prenecrotic mdx muscle despite the absence of dystrophin and that AQP4 is lost after the onset of muscle degeneration. Analysis of various dystrophin transgenic mice reveals that AQP4 is lost even when the dystrophin-glycoprotein complex is present, suggesting that loss of AQP4 is not directly resulting from loss of the DGC. AQP4 was also lost in muscular dystrophies caused by primary mutations in the sarcoglycan genes. Taken together, our data demonstrate that AQP4 loss in skeletal muscle correlates with muscular dystrophy and is a common feature of pathogenesis.-Crosbie, R. H., Dovico, S. A., Flanagan, J. D., Chamberlain, J. S., Ownby, C. L., Campbell, K. P. Characterization of aquaporin-4 in muscle and muscular dystrophy.


Key Words: aquaporin • water channel • dystrophin • sarcoglycans • DMD


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MUSCULAR DYSTROPHIES REPRESENT a group of progressive muscle disorders characterized by extensive muscle wasting and weakness. Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene that result in loss of dystrophin, a peripheral membrane protein localized to the sarcolemma through its association with the dystrophin-glycoprotein complex (DGC) (1 2 3 4 5) . The DGC components consist of the sarcoglycan-sarcospan subcomplex, {alpha}-/ß-dystroglycan, the syntrophins, and dystrobrevin (reviewed in ref 6 ). The sarcoglycans are composed of {alpha}-, ß-, {gamma}-, and {delta}-sarcoglycan, which are transmembrane glycoproteins (reviewed in ref 6 ). Sarcospan, a four-transmembrane protein with tetraspanin-like characteristics, is tightly associated with the sarcoglycans (7 , 8) . Dystrophin binds to filamentous actin and the intracellular portion of ß-dystroglycan (10 11 12 13) . {alpha}-Dystroglycan is a high-affinity receptor for several extracellular matrix components, including laminin and perlecan (4 , 9 10 11 12 13) . Dystrobrevin is a cytosolic protein associated with the syntrophins and dystrophin (18 19 20 21) . Together, the DGC functions to structurally link the extracellular matrix with the intracellular actin network (14) . In DMD, loss of dystrophin results in loss of the entire DGC complex, which renders the muscle plasma membrane susceptible to contraction-induced damage (14 , 15) .

Mutations in {alpha}-, ß-, {gamma}-, or {delta}-sarcoglycan result in autosomal recessive limb girdle muscular dystrophy (AR-LGMD) types 2D, 2E, 2C, and 2F (24 25 26 27 28 29 30 31 32) . Loss of one sarcoglycan generally results in absence of the entire sarcoglycan-sarcospan subcomplex, although cases of partial sarcoglycan loss have been reported (16) . Mouse models for DMD and AR-LGMD have shed light on some of the pathogenetic mechanisms that contribute to muscular dystrophy. For instance, comparisons of the {alpha}-, ß-, and {delta}-sarcoglycan knockout mice have revealed that ß- and {delta}-sarcoglycan null mice have a higher incidence of cardiomyopathy than do {alpha}-sarcoglycan knockout mice (17 18 19) . Cardiomyopathy likely develops from microvascular constrictions of the blood vessels in {delta}- and ß-sarcoglycan knockouts due to the loss of the sarcoglycan-sarcospan complex within the smooth muscle vasculature (18 , 19) .

To determine factors that contribute to disease pathogenesis, much effort has been made to identify proteins that are affected during muscle degeneration. Recent studies have shown that aquaporin-4 (AQP4) is absent in the mdx mouse, a model for DMD (20 , 21) . AQP4 is a mercury-insensitive water channel that regulates flux of H2O molecules across the membrane bilayer in response to changes in osmolarity within a cell (22) . AQP4 is preferentially expressed in fast twitch muscle, which has higher water permeability than that of slow twitch fibers (20) . AQP4 forms oligomers at the plasma membrane, which are observed as orthogonally aggregated particles (OAPs) of 6–7 nm in freeze fracture electron microscopy (23 , 24) . OAP structures are nearly absent in membranes of DMD patients and the mdx mouse (25) , which is consistent with loss of AQP4 from dystrophin-deficient muscle.

Aquaporins may play an important role in regulating the rapid changes in myofiber volume during muscle contraction (26 , 27) . Muscle cells accommodate large changes in hydrostatic forces as well as the intracellular generation of osmotically active solutes from lactate production and metabolism of creatine phosphate (28) . In the present study, we investigate the expression of AQP4 in muscle and in several animal models of muscular dystrophy. We report that AQP4 is lost in dystrophin- and sarcoglycan-deficient animal models of muscular dystrophy. Furthermore, we provide evidence that reduced AQP4 levels are a common feature of muscle pathology regardless of the primary cause of muscle degeneration.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Animal models
Wild-type (C57BL/10) and mdx (C57BL/10ScSn) mice were obtained from Jackson Laboratories (Bar Harbor, ME). Dystrophin transgenic mice have been described (29 30 31 32 33) . Male F1B and BIO 14.6 cardiomyopathic hamsters were obtained from BioBreeders (Fitchburg, MA). Generation and characterization of {alpha}- (Sgca null), {delta}- (Sgcd null), and ß-SG-deficient mice (Sgcb null) have been reported before (17 18 19) . All mice used in experiments were between 6 and 15 wk of age unless otherwise indicated. All animals were maintained at the University of Iowa Animal Care Unit in accordance with animal usage guidelines.

Antibodies and immunoblotting
Polyclonal antibodies to AQP4 were purchased from Alpha Diagnostics (AQP41, San Antonio, TX). Affinity-purified polyclonal aquaporin antibodies were also purchased from Chemicon (AB3068, Temecula, CA). Affinity-purified antibodies to SSPN (Rabbit 236) have been described (7) . KCl-washed skeletal muscle microsomes were prepared from adult mice, as described previously (7) . Protein samples (75 µg/lane) were resolved under reducing conditions by 3–15% SDS-PAGE and transferred to PVDF (Immobilon-P) membranes (Millipore Corp., Bedford, MA). PVDF membranes were probed with anti-AQP4 antibodies (Chemicon) at a dilution 1:200. After incubation with primary antibody, blots were probed with HRP-conjugated secondary antibodies and developed using enhanced chemiluminescence (Supersignal; Pierce Chemical Co., Rockford, IL).

Immunofluorescence
Transverse muscle cryosections (7 µm) were analyzed by immunofluorescence as described (7) . Aquaporin antibodies were incubated at a dilution of 1:500 with mouse muscle cryosections. After washing with Tris-buffered saline (TBS; 10 mM Tris-HCl, 150 mM NaCl, pH 7.4), the sections were incubated with either Cy3- or FITC-conjugated secondary antibodies at a dilution of 1:250 (Jackson Immunoresearch, West Grove, PA) for 1 h at room temperature. For staining of neuromuscular junctions, samples were simultaneously incubated with fluorescein-conjugated {alpha}-bungarotoxin (1:1000; Molecular Probes, Eugene, OR). After washing with TBS, the slides were mounted with Vectashield mounting medium (Vector Laboratories, Burlingame, CA) and observed under a Bio-Rad MRC-600 laser scanning confocal microscope (Hercules, CA). Digitized images were captured under identical conditions.

Recombinant adenovirus injections
Preparation of the {delta}-SG recombinant adenovirus and intramuscular injections were performed as described previously (34) . Briefly, 109 viral particles in 100 µl of normal saline were injected into the quadriceps femoris of 3-wk-old BIO 14.6 hamsters after the animals were anesthetized by intraperitoneal injection of sodium pentobarbital (Nembutal, Abbott Laboratories, Chicago, IL) at a calculated dose of 75 mg/kg. Quadriceps muscle was collected 2 wk after the injection.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The aquaporins constitute a family of channels that transport water molecules across a cellular membrane. Aquaporins have six transmembrane regions that traverse the plasma membrane and two functional loops, which are also embedded in the bilayer (reviewed in ref 35 ). Eleven aquaporin proteins have been identified from a variety of mammalian tissues. Previous reports demonstrating that AQP4 is absent in dystrophin-deficient skeletal muscle (20 , 36 , 37) prompted our use of other murine models to investigate the fate of AQP4 during muscular dystrophy. Dystrophic muscle is characterized by elevated Ca2+ levels, sarcolemma damage, central nucleation, and a large variation in fiber size (Fig. 1 ).



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  		Figure 1. Muscle degeneration in mdx mice. Schematic representation of transverse skeletal muscle cross sections from dystrophin-deficient mdx mice that possess a premature stop codon within exon 23 of the dystrophin gene. From birth to ~3 wk of age, mdx muscle appears histologically normal although the DGC complex is absent (40) . Myofibers are of uniform size with nuclei located at the subsarcolemma. At 3 wk of age, myofibers exhibit an increase in intracellular Ca2+ as the first signs of myofiber necrosis and membrane damage appear. Fibers show a greater variation in size with many hypertrophic fibers. Regeneration results in new myofibers with nuclei located in the center of the cell. Cycles of necrosis and regeneration eventually lead to fibrosis and infiltration of immune cells.

Membrane localization of AQP4 in multiple models of dystrophin deficiency
Chamberlain and colleagues have shown that transgenic expression of various dystrophin isoforms in muscle can restore membrane localization of the DGC and subsequently prevent onset of muscular dystrophy. The transgenes, which were individually expressed on an mdx background, encode truncated dystrophin products (29 , 31 , 33 , 38) . Muscle from the transgenic mice was previously evaluated for its morphology and for the ability of the transgene to restore sarcolemma localization of the DGC (29 , 31 , 33 , 38) . We detected AQP4 expression in skeletal muscle cross sections by indirect immunofluorescence staining with AQP4 antibodies and found normal levels of expression in muscle from adult {Delta}4–7 ({Delta}ABD), {Delta}71-74 ({Delta}330), and {Delta}75–78 mice (Fig. 2 ). Dystrophin transgenes in these mice restored membrane localization of the DGC and prevented symptoms associated with muscle degeneration. However, AQP4 was dramatically reduced in muscle from {Delta}1–62 (Dp71) and {Delta}17–48 (Becker) mice (Fig. 2) . The DGC complex is present in {Delta}1–62 (Dp71) and {Delta}17–48 mice, suggesting that loss of APQ4 is not directly resulting from loss of the DGC. Rather, the absence of AQP4 correlates with even mild dystrophy (29 , 31 , 33 , 38) .



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  		Figure 2. Restoration of AQP4 by dystrophin transgenic products. Various truncated and internally deleted dystrophin transgenes were expressed on an mdx background and previously analyzed for their ability to rescue the mdx phenotype. Skeletal muscle cryosections from adult ‘Becker’ ({Delta}17–48), {Delta}1–62 (Dp71), {Delta}ABD (ABD), {Delta}71–74 ({Delta}330), and {Delta}75–78 mice were stained with AQP4 antibodies and visualized by indirect immunofluorescence. Muscle pathology is present in Dp71 and {Delta}17–48 transgenic mice, as previously documented (29 , 31 , 33 , 38) . Bar, 50 µm.

AQP4 is expressed in young, prenecrotic mdx mice
To distinguish whether loss of AQP4 from transgenic mice was the result of direct interaction with dystrophin or a consequence of muscle pathology, we examined muscle from mdx mice at various stages of muscle degeneration. The dystrophic process in dystrophin-deficient mdx mice is evident at 3 wk of age (Fig. 1) . During the prenecrotic stage (from birth to ~3 wk), muscle histology is normal despite the loss of dystrophin (39) . The cycles of myofiber necrosis and regeneration as well as increased membrane fragility are evident in mdx mice just after 3 wk. It is not clear why normal muscle morphology exists until this time, given that the DGC is absent in mdx mice from birth (40) . Quadriceps muscle from wild-type and mdx mice were examined for expression of AQP4. We found that prenecrotic mdx mice ages 1 and 2 wk had normal immunostaining of AQP4 at the sarcolemma (Fig. 3 ), despite absence of the DGC. Expression of AQP4 in 3-wk-old mdx muscle is heterogeneous, as seen by the absence of AQP4 in some, but not all myofibers. Older (>12 wk) mice completely lacked sarcolemma expression of AQP4. Wild-type mice of all ages had normal, high levels of AQP4 expression (Fig. 3) .



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  		Figure 3. AQP4 membrane localization is normal in young mdx mice. Sections from quadriceps femoris muscles of wild-type and mdx mice at ages 1, 2, and 3 wk were stained with AQP4 antibodies. AQP4 staining was visualized by indirect immunofluorescence with Cy3-conjugated secondary antibodies. AQP4 expression decreased in mdx muscle with the onset of muscle degeneration at 3 wk of age. Sarcolemma localization of AQP4 was not affected by age in wild-type muscle. Bar, 50 µm.

AQP4 in sarcoglycan-deficient models of muscle damage
Mice with targeted deletions in the SG genes were used to determine whether AQP4 expression is affected in sarcoglycan-deficient models of limb girdle muscular dystrophy. Mutations within {alpha}-, ß-, {gamma}-, and {delta}-sarcoglycan are responsible for causing autosomal recessive limb girdle muscular dystrophy types 2D, 2E, 2C, and 2F (24 25 26 27 28 29 30 31 32) . We specifically examined muscle from adult (>8 wk) {alpha}-, ß-, and {delta}-SG null mice (Scga, Sgcb, and Sgcd), which lack the SG-SSPN subcomplex (8 , 17 18 19) . AQP4 expression is severely diminished in each of these mice over 8 wk of age (Fig. 4 A). Identical to young, prenecrotic mdx mice, young (<3 wk) sarcoglycan-deficient mice exhibit normal expression of AQP4 (data not shown). As further demonstration of the loss of AQP4 from the sarcolemma of sarcoglycan-deficient muscle, we immunoblotted KCl-washed membranes prepared from skeletal muscle of adult wild-type (wt), mdx, Sgca, and Sgcd null mice. AQP4 is present in wt samples but absent in Sgca and Sgcd membranes (Fig. 4B ). Skeletal muscle membranes from mdx mice are shown for comparison.



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  		Figure 4. AQP4 localization sarcoglycan-deficient muscular dystrophy. A) Cryosections from quadriceps of adult wild-type (wt), {alpha}-, ß-, and {delta}-SG null mice (Scga, Scgb, Scgd) were stained with antibodies to AQP4. Sections were visualized by indirect immunofluorescence with Cy3-conjugated secondary antibodies. B) Skeletal muscle membranes were prepared from adult wild-type (wt), mdx, {alpha}-, and {delta}-SG null mice were analyzed by 3–15% SDS-PAGE and immunoblotted using antibodies against AQP4. AQP4 migrates at ~32 kDa. The level of AQP4 expression is dramatically reduced in the sarcoglycan and dystrophin-deficient muscle. Molecular weights are indicated on the left (x103 Da).

The BIO 14.6 hamster represents another model for {delta}-SG-deficient limb girdle muscular dystrophy-2F with cardiomyopathy (41 , 42) . A large deletion of the {delta}-SG gene (43 , 44) causes selective loss of the entire SG-SSPN subcomplex from skeletal muscle of the BIO 14.6 hamster (16 , 17 , 42 , 45 , 46) . Expression levels of ß-DG and dystrophin are not altered by loss of the SG-SSPN complex (42 , 46) . We now show that AQP4 expression is also absent from the BIO 14.6 sarcolemma in adult hamsters (Fig. 5 ). AQP4 expression is maintained within the BIO 14.6 muscle sarcolemma after delivery of an adenovirus encoding {delta}-SG (Fig. 5) . Injection of the {delta}-SG adenovirus into young BIO 14.6 hamsters maintains expression of the SG-SSPN subcomplex and prevents the pathogenesis of muscular dystrophy (8 , 34) .



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  		Figure 5. Restoration of AQP4 expression in sarcoglycan-deficient muscle. Loss of aquaporin from the {delta}-sarcoglycan-deficient BIO 14.6 hamster can be restored with recombinant {delta}-SG adenovirus. Quadriceps muscle of the BIO 14.6 hamster was injected with 109 particles of {delta}-SG adenovirus particles (BIO 14.6+{gamma}-SG). Tissue was harvested 7 days postinjection. Muscle cryosections from these injected animals as well as from the F1B and BIO 14.6 (uninjected) hamsters were examined for aquaporin expression by indirect immunofluorescence with AQP4 antibodies. Bar, 50 µm.

AQP4 is not localized to the neuromuscular junction
In our survey of AQP4 expression in skeletal muscle, we discovered that AQP4 was only expressed extrasynaptically. To visualize neuromuscular junctions (NMJs), cryosections from the quadriceps femoris of wild-type mice were stained with fluorescein {alpha}-bungarotoxin, which selectively binds to acetylcholine receptors at the NMJ (Fig. 6 ). By indirect immunofluorescence, we showed that AQP4 is expressed at the sarcolemma but not at the NMJ (Fig. 6) . In fact, we never observed AQP4 at the sarcolemma adjacent to innervated regions of myofibers.



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  		Figure 6. AQP4 is excluded from the neuromuscular junction. Analysis of aquaporins at the neuromuscular junction of adult wild-type murine muscle. AQP4 was visualized by indirect immunofluorescence with Cy3-conjugated secondary antibodies (AQP4, red) and synaptic sites were identified by fluorescein-{alpha}-bungarotoxin staining (BTX, green). Merged image (overlapping sites are yellow) is shown in the right panel. AQP4 is not localized to the NMJ and is clearly excluded from synaptic regions. Bar, 50 µm.

Enrichment at the NMJ is a common characteristic of proteins associated with the dystrophin-glycoprotein complex. At the NMJ, dystrophin is replaced by a structurally and functionally similar protein, utrophin (47 48 49 50 51 52) . The demonstration that AQP4 is not found in NMJs is a clear indicator that AQP4 is not associated with the utrophin-glycoprotein complex. Furthermore, our data suggest that therapeutic up-regulation of utrophin in dystrophin-deficient muscle would not restore AQP4 to the sarcolemma membrane.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
We show that AQP4 is deficient in six distinct animal models of muscular dystrophy. Our data demonstrate that loss of AQP4 in dystrophic muscle is a consequence of muscle pathology. This is emphasized by the normal expression of AQP4 in young, prenecrotic dystrophin-deficient and sarcoglycan-deficient muscle, despite the absence of syntrophins and other DGC components (Fig. 3 ; data not shown). It is not clear whether loss of aquaporin during muscle degeneration contributes to the progress of disease pathogenesis. We also find normal expression of AQP4 in nNOS null mice (53) and sarcospan-deficient mice (54), which have normal DGC expression and muscle morphology (data not shown). A recent report demonstrates that protein and mRNA levels of AQP4 are diminished in denervated muscle, suggesting that innervation may also influence AQP4 expression (55) . Hind limb suspension increased AQP4 expression in rat soleus, suggesting that AQP4 levels may be determined by the glycolytic capabilities of muscle fibers (37) .

It is interesting to speculate on the possible roles of AQP4 within distinct muscle substructures. Measurements of force generation and osmostic water permeability of EDL muscles from AQP4-/- and mdx mice suggest that AQP4 null mice are physiologically normal (24) . The authors conclude that AQP4 does not play an important role in regulating water homeostasis in either normal or dystrophic muscle (24) . Compensation for aquaporin function by an undetermined water channel may at least partially explain results from the AQP4 null mice. However, results from AQP4-/- mice do not agree with the well-established fragility and osmotic instability of the dystrophin-deficient sarcolemma (14 , 15 , 56) . Aquaporins are poised to play an important role in physiological water homeostasis in muscle, given that myofibers must rapidly respond to changes in cell volume during muscle contraction. Our data clearly demonstrate that loss of aquaporins is a common feature of skeletal muscle pathology. However, it is unclear whether loss of aquaporins in muscular dystrophy contributes to muscle pathology. This could be tested directly by generating mdx:AQP4 double null mice. Young double mutant mice (<3 wk of age) lacking both dystrophin and AQP4 may exhibit earlier symptoms of muscular dystrophy, indicating that loss of AQP4 contributes to muscle pathology. Recent analysis of skeletal muscle has demonstrated that AQP4 is enriched in type IIA and B (fast) myofibers and that type IIB fibers are the first fiber type to exhibit reduction of AQP4 in mdx muscle (36 , 37) . Type IIB fibers are preferentially affected in DMD patients (57) . The authors reported that mRNA levels of AQP4 are not reduced in dystrophin-deficient muscle, suggesting that loss of AQP4 protein occurs post-transcriptionally (36 , 37) .

Yokota and colleagues have reported that AQP4 is absent in skeletal muscle and perivascular astrocyte end feet of {alpha}1-syntrophin null mice, indicating that this syntrophin isoform plays a role in either targeting or stabilizing AQP4 at the sarcolemma (58) . Syntrophins interact directly with dystrophin and mediate the attachment of dystrobrevin and nNOS to the DGC complex (59 , 60) . Recently, Neely and colleagues demonstrated that AQP4 could be immunoprecipitated with syntrophin and ß-dystroglycan from chemically cross-linked brain and skeletal muscle lysates (61) . Syntrophin expression is normal in sarcoglycan-deficient mice and the BIO14.6 hamster (data not shown). Muscle from the {alpha}1-syntrophin knockout mice appears normal and does not display any signs of muscle damage (62) .

Taken together, our data demonstrate that AQP4 is lost from dystrophic muscle as a secondary consequence of muscle pathology. The molecular event that triggers the reduction of aquaporins in muscle disease is unknown, but we speculate that it is an early event in pathogenesis. For instance, aquaporins may be very sensitive to increases in intracellular Ca+2 levels, which may activate proteases leading to premature aquaporin protein degradation. Understanding the mechanisms of muscle pathogenesis will provide insights for therapies aimed at blocking or preventing the onset of degeneration.


   ACKNOWLEDGMENTS
 
We thank the University of Iowa Diabetes and Endocrinology Research Center (NIH DK25295). We are indebted to Beverly L. Davidson (University of Iowa) and the University of Iowa Gene Transfer Vector Core (supported in part by the Carver Foundation). We also thank L. E. Lim (University of Iowa) for adenoviral injected BIO 14.6 muscle samples. We are grateful to M. Durbeej, F. Duclos, and R. Coral-Vazquez for the Sgcb, Sgca, and Sgcd mice. We thank T. R. Colberg for outstanding technical support in isolating ACL myotoxin and S. Lowen and R. Barresi for help with the manuscript. We also thank R.D. Cohn for myotoxin injections. R.H.C. is supported by the Robert G. Sampson postdoctoral research fellowship from the Muscular Dystrophy Association. This work was supported by grants from the National Institutes of Health (5R01AI26923 to C.L.O. and AR44533 to J.S.C.) and from the College of Veterinary Medicine, Oklahoma State University (C.L.O.). This research was also supported by a grant from the Muscular Dystrophy Association (K.P.C. and J.R.S.). K.P.C. is an Investigator of the Howard Hughes Medical Institute.


   FOOTNOTES
 
1 Current address: Department of Physiological Sciences, Molecular Biology Institute, 621 Charles E. Young Drive South, University of California, Los Angeles, CA 90095, USA.

Received for publication April 26, 2001. Revision received March 15, 2002.
   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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