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(Circulation Research. 2003;92:554.)
© 2003 American Heart Association, Inc.

Integrative Physiology

Vasomodulation by Skeletal Muscle–Derived Nitric Oxide Requires -Syntrophin–Mediated Sarcolemmal Localization of Neuronal Nitric Oxide Synthase

Gail D. Thomas, Philip W. Shaul, Ivan S. Yuhanna, Stanley C. Froehner, Marvin E. Adams

From the Departments of Internal Medicine (G.D.T.) and Pediatrics (P.W.S., I.S.Y.), University of Texas Southwestern Medical Center, Dallas, Tex, and Department of Physiology and Biophysics (S.C.F., M.E.A.), University of Washington, Seattle, Wash.

Correspondence to Gail D. Thomas, Department of Internal Medicine, Division of Hypertension, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd, Dallas, TX 75390-8586. E-mail gail.thomas{at}utsouthwestern.edu

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Neuronal nitric oxide synthase (nNOS) is abundantly expressed in skeletal muscle where it associates with the dystrophin complex at the sarcolemma by binding to the PDZ domain of -syntrophin. Nitric oxide (NO) produced by skeletal muscle nNOS is proposed to regulate blood flow in exercising muscle by diffusing from the skeletal muscle fibers to the nearby microvessels where it attenuates -adrenergic vasoconstriction. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the vasoregulatory effect of skeletal muscle–derived NO. To test this hypothesis, we performed experiments in -syntrophin null mice and in transgenic mice expressing a mutated -syntrophin lacking the PDZ domain (PDZ), both of which are characterized by reduced sarcolemmal nNOS. We found that modulation of -adrenergic vasoconstriction was greatly impaired in the contracting muscles of the -syntrophin null mice and transgenic PDZ mice compared with wild-type mice and transgenic mice expressing full-length -syntrophin. These in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the dystrophin-associated protein -syntrophin and may have implications for the development of potential gene therapy strategies to treat muscular dystrophy or other muscle-related diseases.

Key Words: -adrenergic • muscle contraction • blood flow • -syntrophin • PDZ domain

	Introduction

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Human muscular dystrophies are a heterogenous group of progressive, often lethal, diseases for which there currently are no established cures and few therapeutic options. The most common and one of the most severe forms of muscular dystrophy, Duchenne muscular dystrophy (DMD), is caused by mutation in the gene encoding the cytoskeletal protein dystrophin.¹ Associated with dystrophin at the sarcolemma is a highly organized complex of transmembrane proteins that include the sarcoglycans, dystroglycans, and sarcospan and peripheral membrane cytoskeletal proteins such as the syntrophins, the dystrobrevins, and cortical actin. Mutations in the genes encoding sarcoglycans, dystroglycans, or dystrobrevin also result in muscular dystrophy.^2–5 Despite a wealth of new information about the molecular basis of the muscular dystrophies, relatively little is known about how these primary genetic defects translate into a dystrophic phenotype.

One of the proposed functions of the dystrophin complex is to maintain the structural integrity of the muscle cell membrane.⁶ Disruption of the complex, which occurs in DMD, is thought to lead to sarcolemmal instability, rendering the muscle cells more susceptible to mechanical or contraction-induced damage.⁷ However, the presence of nonstructural proteins within the dystrophin complex and the generation of mouse models that develop muscular dystrophy without evidence of mechanical injury⁸ or with minimal physical disruption of the dystrophin complex⁴ suggest that the proteins of the dystrophin complex individually or collectively may subserve additional functions. Because numerous signaling molecules have been found to be associated with the dystrophin complex,⁹ one such function may be the formation of transmembrane signaling cascades involved in sensing and responding to changes in the mechanical or metabolic activity of the muscle fibers.

One of the signaling molecules identified as a component of the dystrophin complex is a muscle-specific splice variant of the neuronal isoform of nitric oxide synthase (nNOS),^10,11 which is recruited to the sarcolemma by binding to the dystrophin-associated protein -syntrophin.^12,13 Although abundantly expressed in skeletal muscle, the precise function of nNOS is incompletely understood. Skeletal muscle– derived nitric oxide (NO) has been implicated in myocyte differentiation, excitation-contraction coupling, glucose uptake, cellular respiration, redox regulation, and gene expression.¹⁴ In addition, recent studies in experimental mouse models and in humans suggest that skeletal muscle–derived NO plays an important role in the regulation of blood flow in exercising muscle by modulating -adrenergic vasoconstriction.^15–17 Such modulation is defective in the contracting skeletal muscle of nNOS null mice,^16,17 DMD patients,¹⁵ and mdx mice,¹⁶ an animal model for DMD that harbors a mutation in the dystrophin gene. The dystrophin-deficient skeletal muscle of mdx mice and DMD patients also exhibits a large reduction of both sarcolemmal and soluble nNOS,^10,11 suggesting that the loss of NO signaling might be responsible for the observed impairment in vascular regulation. In fact, transgenic expression of nNOS at high levels in the mdx mouse dramatically improves the skeletal muscle pathology.¹⁸ However, the mechanism by which nNOS protects muscle from degeneration is not known.¹⁹

Although previous studies identified a novel vasoregulatory function of NO generated by nNOS in skeletal muscle, a key unanswered question is whether the cellular localization of nNOS is important for the appropriate transduction of the NO signal from the skeletal muscle fibers to the vasculature. In the present study, we hypothesized that sarcolemmal localization of nNOS is a critical determinant of the ability of skeletal muscle–derived NO to exert its vascular effects. To test this hypothesis in vivo, we performed experiments in -syntrophin null mice and in transgenic mice expressing a mutated -syntrophin lacking the PDZ domain (PDZ), both of which are characterized by a selective reduction of sarcolemmal nNOS.^20–22 These mice are ideal models to examine the functional relevance of sarcolemmal targeting of nNOS because the dystrophin complex is largely intact. In addition, the mice do not develop muscular dystrophy, precluding secondary effects of muscle degeneration.^20,21 We found that the normal modulation of -adrenergic vasoconstriction was greatly impaired in the contracting muscles of the -syntrophin null mice and transgenic PDZ mice compared with wild-type mice and transgenic mice expressing full-length -syntrophin. These data demonstrate the functional importance of appropriate membrane targeting of nNOS by -syntrophin and may have implications for the development of potential gene therapy strategies designed to reintroduce nNOS into dystrophic skeletal muscle.

	Materials and Methods

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Mice
We used previously described male and female -syntrophin null (-syn^-/-) mice and their wild-type C57Bl6 controls.²¹ We also used transgenic mice expressing full-length -syntrophin (Tg -syn) or a mutated -syntrophin lacking the PDZ domain (Tg PDZ) on the -syntrophin null background.²² The -syn^-/- mice were backcrossed onto the C57Bl6 strain and the transgenic mice were backcrossed onto either the C57Bl6 or -syn^-/- strain for at least 4 generations. Mice (21 to 53 g) aged 20 to 44 weeks were used in accordance with protocols approved by the Institutional Animal Care and Research Advisory Committee at the University of Texas Southwestern Medical Center.

Hemodynamic Measurements
Mice were anesthetized with a mixture of Telazol and xylazine (7.5 mg/kg and 20 mg/kg, respectively, IP) followed by -chloralose (80 mg/kg, IV, supplemented as needed). Catheters were inserted in a jugular vein for drug infusions and in a carotid artery for arterial blood pressure measurements. The trachea was cannulated and the mice were ventilated with room air and supplemental oxygen. Core temperature was maintained at 37°C with an external heat source. A Doppler crystal embedded in a soft silastic cuff was placed around the left femoral artery to measure changes in blood flow velocity by recording the pulsatile and mean Doppler shifts in kilohertz using a pulsed Doppler velocimeter (model HVPD-20, Crystal Biotech). A third catheter was inserted into the right femoral artery and advanced to the abdominal aorta and was used for intra-arterial hindlimb infusion of norepinephrine. To accommodate for slight variations in the position of this catheter among mice, the dose of norepinephrine was adjusted in each mouse to elicit maximal decreases in femoral blood flow velocity and vascular conductance of 30% to 50% when injected into the resting hindlimb.

Hindlimb Contraction
The left triceps surae muscles were connected to a force transducer (FT-10, Grass Instruments) via the calcaneal tendon. The left sciatic nerve was exposed, covered with mineral oil, and affixed to stimulating electrodes. To produce intermittent, tetanic contractions, the sciatic nerve was stimulated (model S88, Grass Instruments) at 2 to 3 times the motor threshold voltage with 100-ms trains of pulses (100 Hz, 0.2 ms in duration) at a rate of 30 trains/min. Contraction periods of 10 to 20 minutes were separated by rest periods of at least 20 minutes.

Tissue Extraction and Western Blot Analysis
The gastrocnemius muscle was selected for biochemical analysis because it comprises 80% of the triceps surae mass and contains predominantly fast-twitch fibers²³ that abundantly express nNOS²⁴ (the plantaris muscle also contains mainly fast-twitch fibers and comprises 13% of the triceps surae mass, whereas the soleus contains predominantly slow-twitch fibers and comprises 7% of the triceps surae mass²³). Gastrocnemius muscles were excised, frozen in liquid nitrogen, and stored at -80°C for Western blot analysis of nNOS. Muscles were homogenized in 20 vol of 50 mmol/L Tris buffer (pH 7.5) containing 1 µg/mL aprotinin, 2 µg/mL leupeptin, 20 µmol/L tetrahydrobiopterin, 1 mmol/L dithiothreitol, 1 µg/mL pepstatin A, 10 µg/mL soybean trypsin inhibitor, 1 mmol/L benzamidine, 1 mmol/L EDTA, 0.5 mmol/L phenylmethylsulfonyl fluoride, and 1% Triton X-100. Homogenates were centrifuged at 100 000g for 30 minutes at 4°C to separate membrane pellet and soluble fractions. Pellet fractions were resuspended in 20 vol of buffer. Protein concentrations of muscle fractions were determined using a Bio-Rad DC Protein Assay kit. Samples (100 µg) were resolved by SDS/PAGE on a 6% gel and transferred to nitrocellulose. Membranes were incubated overnight at 4°C with a rabbit polyclonal antibody raised against the N-terminus of nNOS (1:5000) and for 1 hour at room temperature with horseradish peroxidase–conjugated goat anti-rabbit antibody. Immunoreactivity was detected by enhanced chemiluminescence and quantified by densitometry.

NOS Enzymatic Assay
NOS enzymatic activity was determined using previously described methods.²⁵ Soluble and membrane pellet fractions of gastrocnemius muscle homogenates were prepared as described above in 5 vol of 50 mmol/L Tris buffer (pH 7.8) containing 10 µg/mL pepstatin A, 10 µg/mL leupeptin, 10 µg/mL aprotinin, 10 µg/mL N-p-tosyl-L-lysine chloromethyl ketone, 20 µmol/L tetrahydrobiopterin, 3.0 mmol/L dithiothreitol, 1.0 mmol/L phenylmethylsulfonyl fluoride, and 10 mmol/L CHAPS. Protein concentrations of muscle fractions were determined by the Bradford method using bovine serum albumin as the standard.²⁶ NOS activity was determined by measuring the conversion of ³H-L-arginine to ³H-L-citrulline. Fifty microliters of sample was added to 50 µL of buffer yielding final reagent concentrations of 2 mmol/L ß-NADPH, 2 µmol/L tetrahydrobiopterin, 10 µmol/L flavin adenine dinucleotide, 10 µmol/L flavin mononucleotide, 0.5 mmol/L CaCl₂ in excess of EDTA, 15 nmol/L calmodulin, 2 µmol/L cold L-arginine, and 2.0 µCi/mL ³H-L-arginine. After incubation at 37°C for 1 hour, the assay was terminated by adding 400 µL of 40 mmol/L HEPES buffer, pH 5.5 with 2 mmol/L EDTA, and 2 mmol/L EGTA. The terminated reactions were applied to 1-mL columns of Dowex AG50WX-8 (Tris form) and eluted with 1 mL of 40 mmol/L HEPES buffer. ³H-L-citrulline was collected in scintillation vials and quantified by liquid scintillation spectroscopy. NOS activity was fully inhibited by 2.0 mmol/L N^G-nitro-L-arginine methyl ester (L-NAME).

Data Analysis and Statistics
Arterial blood pressures and femoral blood flow velocities (Doppler shifts) were sampled continuously at 100 Hz and saved as digital files (PowerLab, AD Instruments). Femoral vascular conductance (kHz/mm Hg) was calculated online as the mean Doppler shift (kHz) divided by mean arterial pressure (mm Hg). Norepinephrine-mediated vasoconstriction was analyzed by measuring the change in femoral vascular conductance expressed as the maximal decrease from baseline (peak response) or as the integrated area below baseline (total response). Although several doses of norepinephrine often were used in each experiment, a single dose was selected for analysis based on the following criteria: injection into the resting hindlimb evoked femoral blood flow velocity and vascular conductance responses that were characterized by (1) a maximal decrease of 30% to 50% and (2) a total duration greater than 30 seconds.

Statistics were performed using analyses of variance with Scheffe’s post hoc tests or paired Student’s t tests where appropriate (StatView 5.0.1, SAS Institute). Differences were considered statistically significant when P<0.05. Results are presented as mean±SE.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Intracellular Distribution of Skeletal Muscle nNOS Is Altered in -syn^-/- Mice and in Transgenic PDZ Mice
In wild-type mice and Tg -syn mice, nNOS immunoreactivity was detected in both the membrane pellet and soluble fractions of gastrocnemius muscle homogenates (Figure 1). In contrast, in -syn^-/- mice and Tg PDZ mice, nNOS immunoreactivity was present in the soluble fraction but was greatly reduced in the pellet fraction (Figure 1). Likewise, NOS enzymatic activity (pmol/mg protein per min) in soluble fractions of muscle homogenates was similar among all 4 groups of mice but was significantly reduced in pellet fractions in the -syn^-/- mice and Tg PDZ mice (Figure 1). The ratio of total NOS activity (pmol/min) recovered in pellet versus soluble fractions was 121% in wild-type, 128% in Tg -syn, 39% in -syn^-/-, and 35% in Tg PDZ mice.

View larger version (27K): [in this window] [in a new window]   Figure 1. Intracellular distribution of nNOS is altered in -syn-/- mice and in Tg PDZ mice. A, Immunoreactivity for nNOS was similar in the soluble (S) and pellet (P) fractions of gastrocnemius muscle homogenates from wild-type (WT) or Tg -syn mice. In contrast, nNOS immunoreactivity was reduced in pellet versus soluble fractions from -syn-/- or Tg PDZ mice. B, Summary data for densitometric analysis of Western blots probed for nNOS immunoreactivity (n5 for each group). *P<0.05 vs WT. C, NOS enzymatic activity was similar in soluble fractions of muscle homogenates in all of the groups. In wild-type and Tg -syn mice, NOS activity was comparable in pellet and soluble fractions. In contrast, NOS activity was reduced in pellet versus soluble fractions in -syn-/- and Tg PDZ mice (n4 for each group). *P<0.05 vs WT.

Norepinephrine-Mediated Vasoconstriction Is Attenuated in Contracting Hindlimb of Wild-Type, but not -syn^-/-, Mice
In wild-type mice, norepinephrine (6.7±1.6 ng) injected into the arterial supply of the resting hindlimb increased blood pressure and decreased femoral blood flow velocity (-34±4%) and vascular conductance (-39±4%) (Figure 2). As expected, hindlimb contraction alone increased femoral blood flow velocity and vascular conductance (Table). When norepinephrine was injected during hindlimb contraction, the decreases in femoral blood flow velocity (-9±4%) and vascular conductance (-11±3%) were greatly attenuated (Figure 2). Norepinephrine-mediated activation of vasodilatory ß-adrenergic receptors was not responsible for this attenuation, because a similar effect of hindlimb contraction was observed in wild-type mice treated with the ß-blocker propranolol (1 mg/kg, IV) (online data supplement, available at http://www.circresaha.org). The effectiveness of ß-blockade in these mice was verified by lack of a chronotropic response to norepinephrine (online data supplement).

View larger version (24K): [in this window] [in a new window]   Figure 2. Modulation of norepinephrine-mediated vasoconstriction is impaired in -syn-/- mice. A, In a wild-type mouse, intra-arterial injection of norepinephrine (NE) into the resting hindlimb increased blood pressure (BP) and decreased femoral blood flow (FBF), indicating vasoconstriction. In contrast, NE-mediated vasoconstriction was greatly attenuated in contracting hindlimb. B, In wild-type mice (n=5), NE-mediated maximal decreases in femoral vascular conductance (FVC) were attenuated in contracting versus resting hindlimb. C, In an -syn-/- mouse, NE elicited similar decreases in FBF in resting and contracting hindlimb, indicating impaired modulation of NE-mediated vasoconstriction. D, In -syn-/- mice (n=10), large NE-mediated maximal decreases in FVC were observed both in resting and contracting hindlimb. In panels B and D, group means are shown as solid circles. *P<0.05 vs rest.

View this table: [in this window] [in a new window]   Table 1. Hemodynamic and Force Measurements at Rest and During Unilateral Hindlimb Contraction

Norepinephrine (6.5±1.5 ng) injected into the resting hindlimb of -syn^-/- mice elicited maximal decreases in femoral blood flow velocity (-37±4%) and vascular conductance (-42±2%) comparable to the responses observed in wild-type mice (Figure 2). The increases in femoral blood flow velocity and vascular conductance in response to hindlimb contraction alone in -syn^-/- mice also were similar to responses in wild-type mice (Table). In contrast to wild-type mice, in -syn^-/- mice norepinephrine-mediated vasoconstriction was not attenuated in the contracting hindlimb (femoral blood flow velocity, -27±6%; conductance, -34±4%) (Figure 2). Similar results for wild-type and -syn^-/- mice were obtained with either moderate or high doses of norepinephrine (online data supplement), and when norepinephrine-mediated vasoconstriction was expressed as integrated, rather than maximal, decreases in femoral vascular conductance (Figure 4).

View larger version (33K): [in this window] [in a new window]   Figure 4. Comparative effects of norepinephrine in contracting versus resting hindlimbs of wild-type, -syn-/-, and Tg -syn mice. Data shown are the ratios of the integrated decreases in femoral vascular conductance (FVC) evoked by norepinephrine in contracting and resting hindlimb. Norepinephrine-mediated vasoconstriction was attenuated similarly in wild-type (n=5) and Tg -syn mice (n=7), whereas such attenuation was not observed in -syn-/- (n=10) and Tg PDZ (n=6) mice. NOS inhibition with L-NAME prevented the attenuation of NE-mediated vasoconstriction in Tg -syn mice (n=5) but had no effect on the already impaired attenuation in Tg PDZ mice (n=4).

The Wild-Type Phenotype Is Restored in Transgenic Mice Expressing Full-Length -Syntrophin
In Tg -syn mice, norepinephrine (5.0±0.9 ng) injected into the resting hindlimb increased blood pressure and decreased femoral blood flow velocity (-38±3%) and conductance (-44±2%) (Figure 3). As in wild-type mice, norepinephrine-mediated vasoconstriction was greatly attenuated in the contracting hindlimb (femoral blood flow velocity, -11±2%; conductance, -14±1%) (Figure 3). In a subset of these mice (n=5), infusion of the NOS inhibitor L-NAME (10 mg/kg, IV) increased mean arterial pressure (+55±6 mm Hg) and decreased femoral blood flow velocity (-0.45±0.14 kHz) and conductance (-0.008±0.003 kHz/mm Hg) in resting hindlimb. Despite these changes in baseline hemodynamics, norepinephrine injected into the resting hindlimb produced maximal decreases in femoral artery blood flow velocity (-42±6%) and conductance (-41±6%) comparable to the responses elicited before L-NAME. In contrast to the attenuated responses observed before L-NAME, norepinephrine-mediated vasoconstriction was preserved in the contracting hindlimb after L-NAME whether responses were expressed as maximal decreases (femoral blood flow velocity, -37±8%; conductance, -39±6%) or as integrated areas (Figure 4).

View larger version (24K): [in this window] [in a new window]   Figure 3. Modulation of norepinephrine-mediated vasoconstriction is impaired in Tg PDZ mice. A, In a Tg -syn mouse, intra-arterial injection of norepinephrine (NE) into the resting hindlimb increased blood pressure (BP) and decreased femoral blood flow (FBF), indicating vasoconstriction. In contrast, NE-mediated vasoconstriction was greatly attenuated in contracting hindlimb. B, In Tg -syn mice (n=7), NE-mediated maximal decreases in femoral vascular conductance (FVC) were attenuated in contracting versus resting hindlimb. C, In a Tg PDZ mouse, NE elicited similar decreases in FBF in resting and contracting hindlimb, indicating impaired modulation of NE-mediated vasoconstriction. D, In Tg PDZ mice (n=6), large NE-mediated maximal decreases in FVC were observed both in resting and contracting hindlimb. In panels B and D, group means are shown as solid circles. *P<0.05 vs rest.

In Contrast, the Wild-Type Phenotype Is Not Restored in Transgenic Mice Expressing a Mutated -Syntrophin Lacking the PDZ Domain
In Tg PDZ mice, norepinephrine (5.2±0.9 ng) increased blood pressure and decreased femoral blood flow velocity (-34±3%) and conductance (-41±3%) in resting hindlimb (Figure 3). Unlike wild-type mice and Tg -syn mice, but similar to -syn^-/- mice, norepinephrine-mediated vasoconstriction was not attenuated in the contracting hindlimbs of the Tg PDZ mice (femoral blood flow velocity, -26±3%; conductance, -35±2%) (Figure 3). In a subset of these mice (n=4), L-NAME infusion produced the expected increases in mean arterial blood pressure (+64±8 mm Hg) and decreases in femoral blood flow velocity (-0.53±0.06 kHz) and conductance (-0.010±0.001 kHz/mm Hg) in resting hindlimb. After L-NAME, intra-arterial injection of norepinephrine in the resting hindlimb decreased femoral artery blood flow velocity (-43±3%) and conductance (-43±3%). In contracting hindlimb, L-NAME had no further effect on the already impaired modulation of norepinephrine-mediated vasoconstriction whether responses were expressed as maximal decreases (femoral blood flow velocity, -40±3%; conductance, -40±3%) or as integrated areas (Figure 4).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
Skeletal muscle nNOS is reported to play a role in the regulation of a number of cellular processes such as contractile activity,²⁴ glucose uptake,²⁷ and blood flow distribution,¹⁶ but the importance of sarcolemmal targeting of nNOS for these regulatory functions is largely unknown. The principal new finding of the present study is that the normal vasoregulatory function of skeletal muscle–derived NO depends on sarcolemmal localization of nNOS. Genetically engineered mice with greatly reduced sarcolemmal nNOS yet preserved soluble nNOS display impaired NO-dependent modulation of -adrenergic vasoconstriction in contracting skeletal muscle, a defect that is corrected by the reestablishment of normal levels of sarcolemmal nNOS.

A distinctive feature of nNOS in skeletal muscle is its discrete localization to the sarcolemma in conjunction with a complex of transmembrane and intracellular proteins organized around the cytoskeletal protein dystrophin.^10,11 Recruitment of nNOS to the dystrophin complex is mediated by its binding to the 58-kDa adapter protein -syntrophin, which directly binds dystrophin and -dystrobrevin, another dystrophin-associated protein.^28–31 The N-terminus of nNOS, which is unique among the NOS isoforms, contains a PDZ domain that heterodimerizes with the PDZ domain near the N-terminus of -syntrophin.¹² Recent crystallographic and biochemical evidence indicates that this interaction occurs between a ß-hairpin finger located adjacent to the nNOS PDZ domain and the canonical peptide binding groove of the -syntrophin PDZ domain.^13,32 Such PDZ-mediated interactions appear to be a general mechanism by which membrane-associated proteins are recruited to specific subcellular domains to form complex signaling networks.³³

These recent advances have provided valuable insight into the molecular interactions that target nNOS to the sarcolemma; however, little is known about the functional importance of nNOS localization in skeletal muscle. The -syn^-/- mouse is an attractive animal model to use to address this question because in the absence of -syntrophin sarcolemmal nNOS is reduced whereas soluble nNOS is preserved.^20,21,34 Likewise, the Tg PDZ mouse is characterized by a similar reduction of sarcolemmal nNOS due to the absence of the PDZ domain of -syntrophin, thereby preventing the normal binding of nNOS.²² Because the PDZ domain is not required for -syntrophin to bind to dystrophin, PDZ-less -syntrophin localizes normally to the sarcolemma.²² Neither the -syn^-/- mice nor the Tg PDZ mice develop muscular dystrophy,^20–22 although the -syn^-/- mice have structurally defective neuromuscular junctions characterized by reduced levels of acetylcholine receptors, acetylcholine esterase, and utrophin, an orthologue of dystrophin.²¹ Also, the integral membrane protein aquaporin-4, a water channel normally present at the sarcolemma of fast-twitch muscle fibers, is absent from the sarcolemma in both the -syn^-/- mice and the Tg PDZ mice.²² Despite these abnormalities, the contractile properties of skeletal muscle from -syn^-/- mice are normal as indicated by previous in vitro experiments²⁰ and the present in vivo experiments. In contrast, our experiments demonstrate that one potential functional effect of reduced sarcolemmal nNOS in the -syn^-/- mice and Tg PDZ mice is defective vasomodulation in contracting skeletal muscle.

In addition to nNOS, the inducible and endothelial isoforms of NOS (iNOS and eNOS, respectively) also are potential sources of NO in skeletal muscle. Normally, iNOS is undetectable or expressed at low levels in muscle,³⁵ but its expression is increased in diseases such as chronic heart failure or autoimmune inflammatory myopathies or by exposure to exogenous cytokines or bacterial lipopolysaccharides.¹⁴ In our study, it is unlikely that iNOS plays a major role because (1) inflammation is not a characteristic feature of skeletal muscle in any of the lines of mice used and (2) iNOS activity is Ca²⁺-independent and therefore unlikely to be increased during brief bouts of muscle contraction. In contrast, eNOS is constitutively expressed at high levels in the vascular endothelium and at low levels in the cytosol of skeletal muscle fibers.³⁶ However, we previously reported that the normal modulation of -adrenergic vasoconstriction in contracting muscle is impaired in mouse models in which nNOS but not eNOS is reduced in skeletal muscle (nNOS^-/- mice and mdx mice).¹⁶ The impairment in those mice was not exacerbated by treatment with a NOS inhibitor, unlike its effect in wild-type mice to enhance -adrenergic vasoconstriction in contracting muscle.¹⁶ In contrast, NOS inhibition evoked equivalent increases in basal blood pressure and vascular resistance in resting skeletal muscle of wild-type, nNOS^-/-, and mdx mice, suggesting potent inhibition of eNOS in all three genotypes.¹⁶ Likewise, in transgenic mice expressing full-length or PDZ-less -syntrophin in the present study, NOS inhibition had similar effects on basal hemodynamics, but differential effects on -adrenergic vasoconstriction in contracting muscle. Collectively, these data support the hypothesis that NO produced largely by nNOS in contracting skeletal muscle cells serves as a paracrine regulator of adjacent microvessel sensitivity to -adrenergic vasoconstrictors.

The present study was not designed to address the underlying mechanism by which sarcolemmal localization of nNOS may facilitate the vascular effects of skeletal muscle–derived NO; however, at least two potential explanations merit discussion. First, positioning nNOS at the sarcolemma obviously would minimize the diffusion distance between the site of NO production in the skeletal muscle fibers and its site of action at the vascular myocytes, thereby maximizing the likelihood of physiologically relevant amounts of skeletal muscle–derived NO reaching the microvessels.³⁷ Second, membrane targeting of nNOS may create a microenvironment in which the enzymatic activity of nNOS can be precisely coupled to changes in the contractile activity of the muscle fibers. Organization of such a microdomain could serve to position nNOS in close proximity to regulatory factors such as subsarcolemmal Ca²⁺ fluxes, pools of substrate or cofactors, putative enzyme inhibitors including caveolin-3, PIN (protein inhibitor of nNOS), or other as yet unidentified modulatory proteins. Such a juxtaposition of nNOS and regulatory proteins may serve to facilitate NO signaling in a manner analogous to the proposed regulation of eNOS enzymatic activity by its localization to the caveolae of endothelial cells.³⁸

The altered distribution of subcellular NOS activity in the -syn^-/- mice and Tg PDZ mice that we found in the present study is consistent with data previously reported by Kameya et al²⁰ in an independently generated line of -syn^-/- mice. However, in their study, lack of sarcolemmal nNOS in the -syn^-/- mice had no measurable effects on skeletal muscle contractile function.²⁰ In contrast, in our study selective loss of membrane-bound nNOS in the -syn^-/- mice and Tg PDZ mice was associated with impaired vasomodulation in contracting hindlimb. In the Tg PDZ mice, NOS inhibition had no additional effect on the impaired vasomodulation, despite the presence of abundant soluble nNOS. In concert with these findings, using the same experimental preparation, we previously observed defective vascular regulation in the contracting hindlimb of mdx mice and nNOS^-/- mice,¹⁶ two models in which soluble as well as sarcolemmal nNOS is greatly reduced or absent.^10,11 Taken together, the phenotypic similarities of these genotypically diverse mice suggest that the attenuation of -adrenergic vasoconstriction in contracting skeletal muscle corresponds well with sarcolemmal, but not soluble, nNOS.

In contrast, other functions of skeletal muscle–derived NO may be less dependent on sarcolemmal localization of nNOS. For example, in the study by Kameya et al,²⁰ muscles from wild-type and -syn^-/- mice displayed similar force-frequency relationships, which were shifted to the left after treatment with a selective nNOS inhibitor. Those data suggested that in the -syn^-/- mice, sufficient NO is produced by soluble nNOS to modulate contractile force. Also, Wehling et al¹⁸ have reported that expression of a nNOS transgene in the skeletal muscle of mdx mice restores basal NO release from isolated muscle to wild-type values and ameliorates the muscle pathology typically observed in the mdx mouse, an effect that was associated with reduced muscle inflammation and was largely prevented by pharmacological NOS inhibition. Although the cellular localization of nNOS was not described in the latter study, the prediction would be that nNOS would remain largely soluble because of the lack of dystrophin in the mdx mice.

Based on our data showing that mislocalization of skeletal muscle nNOS has a detrimental effect on vascular regulation during electrically evoked muscle contractions in anesthetized mice, we might expect that exercise capacity would be impaired in conscious -syn^-/- mice or Tg PDZ mice. In the same line of -syn^-/- mice that we used in the present study, we previously reported that voluntary wheel-running times and distances were similar in -syn^-/- mice and wild-type controls.²¹ However, voluntary wheel running generally elicits mild cardiovascular responses in rodents.³⁹ Perhaps reduced exercise capacity would be detected in -syn^-/- mice performing more severe exercise, such as forced treadmill running that produces more profound cardiovascular responses mediated in part by activation of the sympathetic nervous system.^39–41 Further investigation of the hemodynamic and sympathetic neural responses to voluntary and forced exercise in these mice is required to address this issue.

Knowledge about the individual molecular components of the dystrophin complex and the physical interactions among these proteins has increased tremendously in recent years, providing new insight into the underlying genetic bases of numerous forms of muscular dystrophy. Much less is known about the physiological and pathophysiological roles of dystrophin and the dystrophin-associated proteins. However, the development of unique animal models such as the -syntrophin null mice and the transgenic -syntrophin mice used in the present study should provide greater insight into the individual and collective functions of the proteins that make up the dystrophin complex. Such information should prove useful for the development of therapeutic strategies to treat muscular dystrophies or other muscle-related diseases. For example, our in vivo mouse studies highlight the functional importance of appropriate membrane targeting of nNOS by the scaffolding protein -syntrophin and suggest that strategies designed to reintroduce nNOS into dystrophic skeletal muscle will restore normal vascular regulation only if nNOS is targeted to the sarcolemma.

	Acknowledgments

 
This work was supported by NIH grants HL06296, NS13345, and HL58888 and the Muscular Dystrophy Association. We thank Ronald G. Victor for his critical review of the manuscript.

Received June 5, 2002; revision received January 7, 2003; accepted January 31, 2003.

	References

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D. W. Wray, P. J. F