1-Syntrophin Gene Disruption Results in the Absence of
Neuronal-type Nitric-oxide Synthase at the Sarcolemma but Does Not
Induce Muscle Degeneration*
Shuhei
Kameya
,
Yuko
Miyagoe
,
Ikuya
Nonaka§,
Takaaki
Ikemoto¶,
Makoto
Endo¶,
Kazunori
Hanaoka
,
Yo-ichi
Nabeshima
**, and
Shin'ichi
Takeda

From the Departments of
Molecular Genetics and
§ Department of Ultrastructural Research, National Institute
of Neuroscience, National Center of Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187-8502, the ¶ Department of
Pharmacology, Saitama Medical School, Moroyama-machi, Saitama 350-04, the
Department of Biosciences, School of Science, Kitasato
University, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, and the
** Institute for Molecular and Cellular Biology, Osaka University, 1-3 Yamada-oka, Suita, Osaka 565, Japan
 |
ABSTRACT |
1-Syntrophin is a member of the family
of dystrophin-associated proteins and is strongly expressed in the
sarcolemma and the neuromuscular junctions. All three syntrophin
isoforms have a PDZ domain that appears to participate in
protein-protein interactions at the plasma membrane.
1-Syntrophin
has additionally been shown to associate with neuronal nitric-oxide
synthase (nNOS) through PDZ domains in vitro. These
observations suggest that
1-syntrophin may work as a modular adaptor
protein that can link nNOS or other signaling enzyme to the sarcolemmal
dystrophin complex. In the sarcolemma, nNOS regulates the homeostasis
of reactive free radical species and may contribute to the oxidative
damage to muscle protein in muscle disease such as Duchenne muscular
dystrophy. In this study, we generated
1-syntrophin knock-out mice
to clarify the interaction between
1-syntrophin and nNOS in the
skeletal muscle. We observed that nNOS, normally expressed in the
sarcolemma, was largely absent from the sarcolemma, but considerably
remained in the cytosol of the knock-out mice. Even though the
distribution of nNOS was altered, the knock-out mice displayed no gross
histological changes in the skeletal muscle. We also discovered that
muscle contractile properties have not been influenced in the knock-out mice.
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INTRODUCTION |
Syntrophins are intracellular peripheral membrane proteins
weighing 58 kDa, originally identified in the postsynaptic membrane of
Torpedo (1). In mammalian skeletal muscle, syntrophins
co-purify with dystrophin, the protein products of the Duchenne
muscular dystrophy (DMD)1
gene (2, 3), and complex with several other dystrophin-associated proteins. The dystrophin complex appears to link the cytoskeleton to
the extracellular matrix in skeletal muscle and stabilize the sarcolemma (4-6).
In human and mouse, three highly conserved but distinct syntrophin
isoforms,
1-,
1-, and
2-syntrophin, are encoded by different genes (7-9). The
1- and
2-syntrophin transcripts are expressed in wide variety of tissues, whereas
1-syntrophin transcript is predominantly expressed in skeletal and cardiac muscle (7, 8). An
immunohistochemical study using specific antibodies revealed that three
syntrophins are concentrated at the neuromuscular junction, but
1-syntrophin is also present at the extrasynaptic sarcolemma (9,
10). The expression of
1-syntrophin at the sarcolemma, however, is
reported to be restricted to fast twitch muscle fibers (9).
All three syntrophins have four protein domains (7); two pleckstrin
homology domains, a PDZ domain, and a carboxyl-terminal syntrophin
unique domain. Pleckstrin homology domain is a small domain containing
less than 100 residues and originally identified as an internally
duplicated motif in pleckstrin (11). It was also revealed in a number
of intracellular signaling and membrane-associated cytoskeletal
proteins. The 57 carboxyl-terminal amino acids of three isoforms, the
syntrophin unique domain (7), are highly conserved and may serve in a
specific interaction with dystrophin and its relatives, dystrobrevin
(8, 12). Finally, the PDZ domain is composed of 90 amino acids and was
originally identified in postsynaptic density-95, discs large, and ZO-1
(13). The PDZ domain is present in diverse families of enzymes and
structural proteins, all of which are concentrated at specialized
cell-cell junctions, such as neuronal synapses, epithelial zona
occludens, and septate junctions. These observations suggest that PDZ
domain may participate in protein-protein interactions at the
plasma membrane (14). Indeed, in skeletal muscle, the association of neuronal nitric-oxide synthase (nNOS) and
1-syntrophin has been demonstrated using a yeast two-hybrid system (15).
1-Syntrophin may
work as a modular adaptor protein that can link a signaling enzyme and
nNOS to the sarcolemmal dystrophin complex.
In DMD and its experimental mouse model, mdx, which lacks
dystrophin expression, nNOS is absent from the sarcolemma and partially accumulates in the cytosol (16). The altered regulation of nNOS in the
dystrophic muscle may augment the toxicity of NO or superoxide and
therefore contribute to myofiber necrosis (17); the derangement of NO
metabolism is responsible for tissue damage in certain diseases (18,
19).
Endogenous NO produced near the sarcolemma has been reported to depress
contractile function (20), although the mechanism of NO action in
muscle contraction is poorly understood. Galler et al. (21)
showed NO-depressed muscle contraction in regard to both mechanical
properties and myofibrillar ATPase activity using the skinned fibers
method. Aghdashi et al. (22) hypothesized that oxidants and
NO interact directly with Ca2+ release channel (ryanodine
receptor 1) in the T-system. The overall magnitude of
cGMP-dependent changes appears to be small (20).
To investigate the role of
1-syntrophin in skeletal muscle and the
interaction between
1-syntrophin and nNOS in vivo, we generated
1-syntrophin knock-out mice. The deficiency of
1-syntrophin in these mice causes the decrease of nNOS expression at
the sarcolemma, indicating the direct interaction of
1-syntrophin
and nNOS in vivo. Very interestingly, the absence of nNOS at
the sarcolemma did not result in an overt degeneration of skeletal
muscle in this mutant mouse. Therefore, the absence of nNOS at the
sarcolemma may not be the major causative factor of muscle degeneration
in DMD or in mdx mice. In addition, the force-frequency
relationship between wild-type and
1-syntrophin knock-out mice
showed no significant difference in muscle contractile properties. We
also examined the effects of the NOS inhibitors, 7-nitroindazole (7-NI)
and nitro L-arginine methyl ester (L-NAME), and
the exogenous NO donor, sodium nitroprusside (NP), on muscle
contraction. Treatment by NOS inhibitors caused the shift of
stimulation-tension curves in the mutant mice, suggesting that muscle
contractile properties of
1-syntrophin knock-out mice are regulated
by the nNOS activity in the cytosol.
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MATERIALS AND METHODS |
Targeting Vector Construction and Transfection of ES
Cells--
The mouse
1-syntrophin gene was isolated from a mouse
genomic library prepared from 129/SvJ mice DNA (Stratagene). It was probed with a 164-base pair RT-PCR product from exon 2 (7). The
targeting vector, as shown in Fig. 1a, consisted of the
6.0-kb genomic DNA containing the 1.2-kb neomycin resistance gene
(BbsI site of exon 2) derived from pMC1neopoly(A) (24). A
1.8-kb herpes simplex virus thymidine kinase gene was attached to the
5' end of the
1-syntrophin-neomycin construction. Finally, the
targeting vector was linearized using the restriction enzyme,
NotI, before transfection by electroporation into E14
embryonic stem cells obtained from the inbred mouse strain 129/SvJ
(23). After detaching the ES cells (107) from their plates
using trypsin, they were suspended in 3.2 ml of cold Hanks' solution
and then mixed with the targeting vector DNA at a concentration of 25 µg/ml in an electroporation cuvette. The electroporation was carried
out at 200 V and 500 microfarads using Gene Pulser (Bio-Rad) apparatus.
Two days following the electroporation, we added 300 µg/ml Neomycin
(G418) to the cultures. On day 4 after electroporation, gancyclovir was
added to the cultures to a concentration of 2 µM to
isolate the cells containing a targeted disruption by positive-negative
selection. Then surviving colonies were cloned after a 10-day selection
period. Homologous recombinants were screened by PCR using the primers
described below. The forward primer (P1), designed from neomycin
resistance gene, was ATTCGCCAATGACAAGAC (24), and the reverse primer
(P2), outside the targeting vector, was CCTCTTTACATCTGTGTCATC. PCR was
carried out for 40 cycles at 95 °C for 1 min, 55 °C for 1 min,
and 72 °C for 2 min by a DNA thermal cycler (Perkin-Elmer).
Generation of Chimeric Mice with Germline Transmission--
We
generated chimeric mice using the blastocyst injection (23) and
aggregation (25) methods. Blastocysts were recovered from a mating of
B6C3F1 (F1 of C57BL/6 and C3H/He) and C57BL/6 mice. ES-injected
blastocysts were transplanted into uteri of pseudopregnant ICR mice.
The resulting chimeric mice were mated with BALB/c mice, and homozygous
mutant mice were generated by an inter-crossing of heterozygotes. The
aggregation method was performed as described previously, with some
modifications to suit our experimental conditions (25). Chimeric mice
were mated with C57BL/6 mice, and homozygous mutant mice were generated
by inter-crossing of heterozygotes.
Genomic Southern Blot Analysis--
We isolated genomic DNA from
ES cells or mouse tails by phenol extraction and ethanol precipitation.
The genomic DNA was digested by EcoRI, fractionated using
electrophoresis through 0.8% agarose gels, and transferred to Hybond
N+ nylon membrane (Amersham Pharmacia Biotech). The blots
were hybridized to the 32P-labeled
NaeI-SpeI fragment of genomic DNA produced using
a Random primer DNA labeling kit (Takara).
Northern Blot Analysis and RT-PCR--
Total RNA was isolated by
using RNAzol B (Tel-Test, Inc.). We purified the poly(A)+
RNA using the mRNA Purification Kit (Amersham Pharmacia Biotech). Poly(A)+ RNA was electrophoresed in a 1.0% denaturing
agarose-formaldehyde gels and transferred to Hybond N+
nylon membrane (Amersham Pharmacia Biotech). The hybrydization was
carried out using 32P-labeled DNA probes constructed by
employing a random primer DNA labeling kit (Takara). The density of the
detected band were calculated using BAS-2000 (Fuji). Mouse
1-syntrophin cDNA (nucleotides 1180-1355) (26) and mouse nNOS
cDNA (nucleotides 1547-2105) (27) were used to detect the mRNA
of
1-syntrophin and nNOS. For RT-PCR, first strand cDNA was
synthesized using random hexamer primers. The forward primer (F1) and
(F2), from exon 1 of
1-syntrophin gene (7) was AGCTGCCAGAAGCGCTGCTG
and GCTGGGCATCAGCATCAAGG, respectively. The reverse primer (R1), from
exon 3 of the
1-syntrophin gene (7) was GAGGTGAGTCCCAGCCAACG.
Western Blot Analysis for
1-Syntrophin--
The total
cellular protein was extracted from mouse hind-limb muscle for Western
blot analysis. We used Bradford method and Coomassie Brilliant Blue
G-250 (Bio-Rad) to determine the protein concentrations. The protein
fractions were extracted with a reducing sample buffer (10% SDS, 70 mM Tris-HCl, 5%
-mercaptoethanol, 10 mM
EDTA). 15 µg protein/lane was separated on a SDS-polyacrylamide gel
(8%). The resulting gel was subsequently transferred to a polyvinylidene difluoride membrane (Millipore) employing an amperage of
242 mA for 2 h. The blot was later incubated with
anti-
1-syntrophin antibody.
1-Syntrophin polyclonal antibody was
prepared by injecting purified synthetic peptides into rabbits
according to standard protocols. Anti-
1-syntrophin antibody was
raised against the peptides CRQPSSPGPQPRNLSEA (amino acids 191-206
(26) of
1-syntrophin plus an amino-terminal cysteine). The antibody
was used at a 1:40 dilution, and its signal was detected using the
enhanced chemiluminescence method (Amersham Pharmacia Biotech) and a
1:5000 dilution of the anti-rabbit secondary antibody (Tago Immunologicals).
Histological Analysis, Immunohistochemistry, and NDP Activity
Assay--
The skeletal muscle of mice were excised postmortem and
then rapidly frozen in liquid nitrogen-cooled isopentane. 6-µm frozen sections were cut and placed on poly-L-lysine-coated
slides. The slides were brought to room temperature, air dried, and
acetone-fixed for 10 min. For histological analysis, the fixed sections
were stained with hematoxylin and eosin. For immunostaining, the
sections were incubated with anti-
1-syntrophin antibody at a
dilution of 1:40 and then stained using fluorescein
isothiocyanate-conjugated anti-rabbit goat antibody (Tago
Immunologicals). An NDP activity assay was performed as described
previously by Kusner and Kaminski (28). For this assay, 10-µm frozen
sections were postfixed in 2% paraformaldehyde in phosphate-buffered
saline, pH 7.4, at 2 h. After rinsing in phosphate-buffered
saline, the sections were incubated in 0.2% Triton X-100 for 10 min at
37 °C. The subsequent reaction was carried out for 1 h in a
dark, humidified chamber at 37 °C in 0.2% Triton X-100, 0.1 mM NADPH, and 0.16 mg/ml nitro blue tetrazolium. The
reaction was terminated by washing with water.
Extraction and Western Blot Analysis for nNOS--
The
extraction of nNOS was performed according to the method described by
Brenman et al. (16). To isolate nNOS, the quadriceps muscle
was homogenized in 10 volumes (w/v) of buffer A (25 mM Tris-HCl, pH 7.4, 100 mM NaCl, 1 mM EDTA, 1 mM EGTA). The nuclei of the muscle were pelleted by
centrifugation at 1000 × g. The supernatant was then
centrifuged at 20,000 × g to yield the supernatant S1.
The resulting heavy microsomal pellet was resuspended in buffer B (500 mM NaCl added to buffer A), incubated for 30 min at 4 °C with agitation, and centrifuged at 15,000 × g,
yielding supernatant S2. The pellet from this last centrifugation was
resuspended in buffer B containing 0.5% Triton X-100, incubated for 30 min at 4 °C with agitation, and centrifuged at 15,000 × g to create supernatant S3 and the final pellet, P. The
fractions were resolved using the sample buffer and analyzed by
SDS-polyacrylamide gel electrophoresis (6% polyacrylamide). The
proteins were transferred to a polyvinylidene difluoride membrane
(Millipore), which was later incubated with anti-nNOS antibody
(Transduction Laboratories).
NOS Catalytic Assays--
NOS catalytic assay were carried out
according to the method described by Brenman et al. (16).
The quadriceps muscle from wild-type, mdx, and homozygous
mutant mice were homogenized in 10 volumes of buffer containing 25 mM Tris-HCl, pH 7.4, 1 mM EDTA, 1 mM EGTA, and 0.1 mM NaCl. The homogenate was
centrifuged at 20,000 × g to reveal the soluble
fraction. The pellet was extracted in the same buffer containing 0.5 M NaCl and centrifuged at 20,000 × g to
create a particulate fraction. To perform the catalytic assay, aliquots
from these fractions were assayed in 125-µl reactions containing
1.8 × 105 cpm of [3H]arginine (53.0 Ci/mmol), 1 mM NADPH, 640 µM
CaCl2, 1 µM calmodulin, 3 µM
each of tetrahydrobiopterin, FAD, and FMN. After incubation for 25 min
at 22 °C, the assays were terminated with 2 ml of 20 mM
HEPES, pH 5.5, 2 mM EDTA. The samples were then applied to 1-ml columns of Dowex AG50WX-8 (Na+ form), which were
eluted with 2 ml of water. [3H]citrulline was quantified
by liquid scintillation spectroscopy of the 4-ml flow-through.
Differences of activity of soluble or particulate fractions between
wild-type and knock-out or mdx mice were calculated using Student's
t test.
Analysis of Contractile Properties of Muscles--
We dissected
the muscle fiber bundles from the diaphragms and extensor digitorum
longus of mice and tied them at both ends with silk filaments. The
bundles were then mounted in a small bath between a pair of stainless
steel rods, one of which was attached to a force transducer. We then
immersed the bundles in physiological salt solution containing: 150 mM NaCl, 4 mM KCl, 2 mM
CaCl2, 1 mM MgCl2, 5 mM
HEPES, 5.6 mM glucose, pH 7.4. Electrical stimulation was
applied through a pair of platinum wires placed on both sides of the
bundles. The stimulus lasted 0.5 ms and measured a voltage of 20 V. The
relative force was calculated by counting the force recorded at a
stimulus frequency of 125 Hz as 100%. In the experiments using bundles
from diaphragm, to assess the effects of NOS inhibitor and NO donor on
contraction, 1 mM 7-NI, 5 mM
L-NAME, or 1 µM NP was added to the
physiological salt solution.
 |
RESULTS |
Generation of
1-Syntrophin Knock-out Mice--
We cloned and
mapped the mouse
1-syntrophin gene and used that information to
disrupt it in the E14 ES cell line, a line derived from the 129/SvJ
mouse strain. Homologous recombination between the targeting vector and
the
1-syntrophin gene resulted in the replacement of the second exon
of the gene with the neomycin resistance gene, thus knocking out the
1-syntrophin gene (Fig. 1a). Five positive clones were
identified from 288 G418- and GANC-resistant clones. Southern blot
analysis using a probe that recognizes an 7.0-kb band in the wild-type
allele and a 3.6-kb band in the targeted allele confirmed our results
(Fig. 1b).

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Fig. 1.
a, restriction map of the endogenous
1-syntrophin gene, the targeting vector, and the properly disrupted
1-syntrophin gene. The herpes simplex virus thymidine kinase gene
(TK) and the neomycin resistance gene (NEO) are
also illustrated. Homologous recombination results in the insertion of
the neomycin resistance gene into the second exon of the
1-syntrophin gene. The location of the probe used for Southern blot
analysis is shown. The probe hybridizes to a 7.0-kb EcoRI
fragment from the endogenous 1-syntrophin allele and to a 3.6-kb
EcoRI fragment from the disrupted gene. b,
Southern blot analysis of EcoRI-digested genomic DNA from
mouse tails.
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Two germline-transmitting mice from the two independently derived E14
ES cell clones were obtained and further studied. Heterozygous animals
identified by Southern blot analysis were bred with each other to
obtain homozygous animals. Heterozygous and homozygous mice are
indistinguishable from wild-type mice and develop normally in nearly
all respects.
1-Syntrophin Expression Is Absent in Homozygous Mutant
Mice--
Our Northern blot analysis revealed the expression of
1-syntrophin mRNA in the skeletal muscle of wild-type mice
through strong signals at 2.2 and 2.6 kb (Fig.
2a). This result is consistent with a previous report regarding
1-syntrophin expression (7). The
size of
1-syntrophin mRNA of heterozygous mutant mice was identical to that of wild-type mice, but the amount of
1-syntrophin mRNA was about 70% of that in wild-type mice. The mRNA of
homozygous mutant mice included a 1.9-kb band and light smears in the
vicinity of 1.5 kb. The amounts were 20% that of wild-type mice.

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Fig. 2.
a, Northern blot analysis of
1-syntrophin mRNA in skeletal muscle of wild-type (+/+),
heterozygous (+/ ), and homozygous mutant ( / ) mice. A broad band
sending a strong signal at 2.2 and 2.6 kb appears in the blots of
wild-type and heterozygous mutant mice. Heterozygous mutant mouse
mRNA demonstrated an approximately 70% density in 1-syntrophin
mRNA compared with wild-type mice. In comparison, homozygous mutant
analysis resulted in a single 1.9-kb band with 20% density compared
with wild-type mice. The positions of size markers are indicated in
kilobase pairs. b, the same filter was probed with -actin
to ensure that equal amounts of mRNA were loaded on the filter.
c, RT-PCR analysis of skeletal muscle mRNAs from
wild-type (lanes 1 and 3) and homozygous mutant
mice (lanes 2 and 4) using the F1 forward primer
and R1 reverse primer (lanes 1 and 2) and the F2
forward primer and R1 reverse primer (lanes 3 and
4). Products at the predicted sizes were obtained from
wild-type mice, whereas shorter products were produced by homozygous
mutant mice. The positions of size markers are indicated in base pairs.
Control RT-PCR amplification of the same mRNA preparations using
primers for 2-syntrophin are also shown (lanes 5 and
6). d, Western blot analysis of skeletal muscle
from wild-type (+/+), heterozygous (+/ ), and homozygous mutant
( / ) mice using anti- 1-syntrophin antibody. Wild-type mice
produced a 59-kDa band from the crude SDS lysate of their skeletal
muscle, whereas homozygous mutant mice produced no detectable band.
Heterozygous mutant mice also produced a 59-kDa band having half the
density of wild-type mice. The positions of the molecular mass markers
are indicated in kilodaltons.
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Our RT-PCR analysis of the skeletal muscle mRNA of wild-type and
homozygous mutant mice employed pairs of primers that spanned exon 1 and 3 of the
1-syntrophin gene. Wild-type mice revealed a PCR
product with an expected size, but homozygous mutant mice had a shorter
than expected product (Fig. 2c). We cloned the PCR product
from homozygous mutant mice and found that the entire exon 2 of the
1-syntrophin gene was missing.
To investigate the expression of
1-syntrophin at the protein level,
we prepared an anti-
1-syntrophin antibody. The polyclonal antibody
was raised against synthetic peptide corresponding to
1-syntrophin
(amino acids 191-206). Using this antibody, we carried out Western
blot analysis of the skeletal muscle lysates of wild-type, heterozygous
mutant, and homozygous mutant mice (Fig. 2d). Wild-type mice
revealed a 59-kDa band that corresponds to
1-syntrophin, whereas
homozygous mutant mice showed no detectable
1-syntrophin. In
addition, we could not detect any truncated fragments that could have
been derived from the exon 2-lacking transcript. Heterozygous mutant
mice produced nearly half the amount of
1-syntrophin compared with
wild-type mice.
Histological and Immunohistochemical Analysis of
1-Syntrophin
Knock-out Mice--
We examined frozen sections of the tibialis
anterior and soleus muscles of wild-type and homozygous mutant mice
using hematoxylin and eosin staining (Fig.
3, a-d). There were no
striking morphologic changes in these skeletal muscles. We could detect
neither degeneration and regeneration of muscle fibers nor cellular
infiltrations and proliferation of connective tissues in the
1-syntrophin knock-out mice. Histochemical analysis using
Myosin-ATPase staining indicated that the differentiation of muscle
fiber types has not been affected.

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Fig. 3.
Histological and Immunohistochemical analyses
of wild-type (a, c, and e) and
1-syntrophin knock-out mice (b, d, and
f). The tibialis anterior (a and
b) and soleus (c and d) muscles of
1-syntrophin knock-out mice showed no obvious differences compared
with those of wild-type mice. Immunohistochemical analyses conducted
using anti- 1-syntrophin polyclonal antibody (e and
f) resulted in sarcolemmal staining principally at the
neuromuscular junctions of wild-type mice, whereas 1-syntrophin
knock-out mouse had a complete loss of staining. Magnification,
×200
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Using anti-
1-syntrophin antibody, we examined adult skeletal muscle
immunohistochemically (Fig. 3, e and f). In
wild-type mice, the expression of
1-syntrophin was detected at the
sarcolemma and was particularly enriched at the neuromuscular junction,
as already reported (10). In homozygous mutant mice, the expression of
1-syntrophin was completely absent in both the sarcolemma and the
neuromuscular junction. The labeling pattern for the
1-syntrophin of
heterozygous mutant mice was indistinguishable from that of wild-type mice.
To investigate the expression of dystrophin-glycoprotein complex in
1-syntrophin knock-out mice, we performed immunohistochemical analysis of adult skeletal muscles using anti-dystrophin and
anti-
-sarcoglycan antibody. The expression of dystrophin and
-sarcoglycan appeared to be not altered in homozygous mutant
skeletal muscle (data not shown).
nNOS Expression in
1-Syntrophin Knock-out Mice--
Recent
research has suggested that
1-syntrophin anchors nNOS to the
sarcolemma (14). We investigated the influence of the loss of
1-syntrophin on the expression of nNOS in the homozygous mutant
muscle. We did this by performing a Northern blot analysis of adult
skeletal muscle (Fig. 4a). In
wild-type mice, a single 10-kb band corresponding to nNOS mRNA was
detected. Homozygous mutant mice produced an equal amount of nNOS
mRNA. In mdx skeletal muscle, the amount of nNOS
mRNA was decreased to 50% in accord with the previous report
(29).

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Fig. 4.
a, Northern blot analysis of nNOS
mRNA in the skeletal muscle of wild-type (+/+), heterozygous
(+/ ), homozygous mutant ( / ), and mdx mice. In all mice
tested, a single 10-kb band appeared. The density of the bands,
compared with wild-type mouse, were 167.5% for heterozygous, 114.4%
for homozygous mutant, and 47.9% for the mdx mice. The
positions of the size markers are indicated in kilobase pairs.
b, the same filter was probed with -actin to standardize
the amounts of RNA loaded on the filter. c and d,
NDP assay of skeletal muscle in wild-type (c) and homozygous
mutant mice (d). NDP activity was restricted to the
sarcolemma in wild-type mice but was completely absent in homozygous
mutant mice. Magnification, ×400. e and f,
subcellular analysis of skeletal muscle in wild-type (e) and
1-syntrophin knock-out (f) mice. Western blot analysis
using anti-nNOS antibody indicated that significant amounts of nNOS
remained in the insoluble pellet (P) even after sequential
extraction of skeletal muscle homogenates with 100 mM NaCl
(S1), 500 mM NaCl (S2), and 0.5%
Triton X-100 (S3) in wild-type mice (e,
upper panel). In 1-syntrophin knock-out mice, nNOS was
largely extracted with 100 mM NaCl (S1) and was
not detected in a pellet (P) fraction (f,
upper panel). Control immunogloblin bands were shown
(e and f, lower panels).
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In DMD and mdx mice, nNOS is absent from the sarcolemma but
expressed in the cytosol although in small amounts. Like nNOS, NDP
activity in skeletal muscle fibers is also concentrated in the
sarcolemma and colocalizes with nNOS (28) (Fig. 4c). In
1-syntrophin knock-out mice, however, NDP activity was completely absent in the sarcolemma (Fig. 4d).
We also investigated the subcellular distribution of nNOS in the
quadriceps muscle of
1-syntrophin knock-out mice (Fig. 4, e and f). In wild-type mice, significant amounts
of nNOS remained in the insoluble pellet (P) even after the sequential
S1, S2, and S3 extractions of skeletal muscle homogenates. By contrast, nNOS was largely removed with 100 mM NaCl (S1) and did not
remain in the pellet (P) fraction in
1-syntrophin knock-out mice.
Our study of nNOS catalytic activity demonstrated that this activity
for nNOS was strongest in the particulate fractions of skeletal muscle
homogenates in wild-type mice in accord with previous report (15)
(Table I). In mdx mice, some
nNOS activity remained in the soluble fractions, whereas activity in
the particulate fractions was decreased. Homozygous mutant has
decreased nNOS activity in the particulate fraction but maintained the
activity in the soluble fractions, compared with wild-type mice. In
addition, the total nNOS activity for homozygous mutant mice was 59%
that of wild-type mice; the activity for mdx mice was only
27% when calculated using the mean value.
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Table I
NOS catalytic assay
nNOS catalytic activity was enriched in the particulate fractions of
skeletal muscle homogenates in wild-type mice. In mdx mice
and homozygous mutant, nNOS activities were decreased in particulate
fractions. Differences between wild-type and knock-out or
mdx mice were calculated by t test. The values
are the means ± S.E. (n = 4 in all comparisons;
×105 cpm mg 1 protein). NS, not significant.
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Contractile Properties of Skeletal Muscles in
1-Syntrophin
Knock-out Mice--
To examine the influence of intracellular
distribution of nNOS in knock-out mice on muscle contraction, we
measured contractile properties of the diaphragm and the extensor
digitorum longus muscles (Fig. 5). We
first measured the relative forces exerted during submaximal
contractions to determine the steady state force-frequency relationship
in the diaphragm of the wild-type and
1-syntrophin knock-out mice.
No significant difference was observed between wild-type and
1-syntrophin knock-out mice at any stimulus frequency measured.
Extensor digitorum longus muscle was also investigated but again did
not produce any differences between wild-type and knock-out mice (data
not shown). In diaphragm, we further investigated the influence of NOS
inhibitors and NO donor on muscle contractions (Fig. 5). Exposure to
the NOS inhibitor 7-NI shifted the force-frequency relationship up and
to the left at both wild-type and
1-syntrophin knock-out mice, and
the relative forces showed no significant difference between the mice.
Exposure to the NOS inhibitor, L-NAME also shifted the
force-frequency relationship up and to the left at both wild-type and
1-syntrophin knock-out mice. However, L-NAME had a
relatively weak effect on contraction in knock-out mice compared with
the effect in wild-type mice. Treatment by the NO donor NP did not
result in any significant changes from the steady state.

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|
Fig. 5.
Force-frequency relationship of the skeletal
muscles of wild-type (a) and 1-syntrophin knock-out mice
(b). The relative force was calculated by counting the
force recorded at a stimulus frequency of 125 Hz as 100%. Means ± S.E. has been shown using symbols and bars. 7-NI caused a
significant shift to the left of the force-frequency relationship in
wild-type mice. L-NAME caused a moderate shift to the left
of the force-frequency relathionship in wild-type muscle. The exogenous
NO donor NP had no effect on contraction in wild-type mice. The
contractile properties of normal, 7-NI-treated and NP-treated
1-syntrophin knock-out mice were not different from wild-type mice.
L-NAME had relatively weak effect on contraction in
1-syntrophin knock-out mice compared with the effect in wild-type
mice. c, the relative force at a stimulus frequency of 60 Hz in
wild-type (+/+) and 1-syntrophin knock-out mice ( / ). Treating
with 7-NI and L-NAME showed statistically significant
effect on contraction both wild-type and knock-out mice when compared
with steady state contraction. Differences of relative force between
NOS inhibitors or donor-treated and steady state were calculated by
t test.
|
|
 |
DISCUSSION |
Our
1-syntrophin knock-out mice produce a truncated transcript
that lacks 186 base pairs of exon 2 of the
1-syntrophin gene. This
missing segment could cause an in-frame deletion and produce a
truncated
1-syntrophin lacking the PDZ domain. However, Western blot
analysis detected no truncated
1-syntrophin in the knock-out mice.
We conclude that a truncated
1-syntrophin probably is unstable and
therefore difficult to detect, although small amount of truncated
1-syntrophin mRNA is identified.
In
1-syntrophin knock-out mice, there is no NDP activity in the
sarcolemma and a considerable amount of nNOS remains at the soluble
fraction. The interaction of
1-syntrophin and nNOS has been
demonstrated in vitro by using the yeast two-hybrid system, pull-down assay, and protein overlay assay (15). Although there is an
altered distribution of nNOS in DMD and mdx mice, it is not
known which protein among the dystrophin-associated proteins is
responsible for the disappearance of nNOS from the sarcolemma. Our data
strongly suggest that
1-syntrophin interacts with nNOS in
vivo via the PDZ domain and anchors nNOS to the submembranous dystrophin complex.
The role of NO in skeletal muscle is not clear. Altering the regulation
of nNOS in dystrophic muscle can augment the toxic interaction of NO
and superoxide with the cell and contribute to myofiber necrosis (17).
In DMD and mdx mice, the preferential degeneration of fast
twitch fiber can be explained by the selective enrichment of nNOS in
fast twitch muscle fibers (20). Our knock-out mice have no obvious
degeneration of their muscle fibers despite the disappearance of
1-syntrophin and nNOS from the sarcolemma. This suggests that the
degeneration process does not arise merely from the altered
distribution of nNOS.
NO produced by nNOS near the sarcolemma of fast twitch muscle fibers
appears to regulate the muscle contraction by opposing the generation
of contractile force (20). Our functional analysis of the contractile
properties of wild-type and
1-syntrophin knock-out mice revealed no
significant changes when tested at steady state. This observation shows
that the muscles of the knock-out mice still retain enough NO to
regulate muscle contraction by depressing the contractile force. This
was also supported by the findings that treatment with a NOS inhibitor,
7-NI, shifted the force-frequency relationship to the left not only in
wild-type mice but also in
1-syntrophin knock-out mice. However,
another NOS inhibitor, L-NAME, had a comparatively weak
effect on muscle contraction of
1-syntrophin knock-out mice than the
effect on that of wild-type mice. We cannot exclude the possibility
that the modest response to L-NAME reflects reduced
expression of nNOS and/or the altered distribution of nNOS in
1-syntrophin knock-out mice. Although Kobzik et al. (20)
showed that the exogenous NO donor, sodium nitroprusside, could depress
the development of tension, our findings reveal that sodium
nitroprusside has no significant effect on contraction. Because the
effect of the exogenous NO donor was relatively modest compared with
the NOS inhibitor in the previous study, the exogenous NO donor might
have a weak effect on contraction in our particular assay. The
biochemical nNOS catalytic activity assay showed that
1-syntrophin
knock-out mice maintained 59% of nNOS activity compared with wild-type
mice and that certain activities were still retained at the cytosol
fraction. We believe that the remaining nNOS activity in the cytosol
regulates the contractile properties of
1-syntrophin knock-out mice.
Although the target of NO in skeletal muscle contraction has not yet
been assigned to either myofibrillar proteins, the Ca2+
release channel, or any other factors, our results suggest that cytosolic nNOS could compensate for the lost activity of
membrane-associated nNOS and provide enough NO to depress the excessive contraction.
1-Syntrophin transcript is also expressed in tissues other than
skeletal and cardiac muscles. The amount of mRNA in tissues other
than striated muscle is very low, and its expression in other tissues
has not yet been fully verified and therefore must be investigated.
Although we have demonstrated an important role for
1-syntrophin in
the function of nNOS within the sarcolemma, further study will be
needed to gain a deeper understanding of the function of
1-syntrophin in the contraction and maintenance of the structure of
skeletal muscle.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Hideo Sugita and Daniel North
for review of this manuscript.
 |
FOOTNOTES |
*
This work was supported by Grant 8A-1 for Nervous and Mental
Disorders from the Ministry of Health and Welfare (to S. T.).The costs of publication of this
article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in
accordance with 18 U.S.C. Section
1734 solely to indicate this fact.

To whom correspondence should be addressed: Dept. of Molecular
Genetics, National Institute of Neuroscience, National Center of
Neurology and Psychiatry, 4-1-1 Ogawa-higashi, Kodaira, Tokyo 187-8502, Japan. Tel. or Fax: 81-42-346-3864; E-mail:
takeda{at}ncnaxp.ncnp.go.jp.
The abbreviations used are:
DMD, Duchenne
muscular dystrophy; NOS, nitric-oxide synthase; nNOS, neuronal NOS; ES, embryonic stem; NDP, nicotinamide adenin dinucleotide
phosphate-diaphorace; 7-NI, 7-nitroindazole; L-NAME, nitro
L-arginine methyl ester; NP, sodium nitroprusside; RT, reverse transcriptase; PCR, polymerase chain reaction; kb, kilobase pair(s).
 |
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