Neuronal Nitric Oxide Synthase Mu Research Articles

Differential superoxide production in phosphorylated neuronal nitric oxide synthase mu and alpha variants

Neuronal nitric oxide synthase (nNOS) is regulated by phosphorylation in vivo, yet the underlying biochemical mechanisms remain unclear, primarily due to difficulty in obtaining milligram quantities of phosphorylated nNOS protein. The functional diversity of the nNOS isoform is also linked to its splice variants. Of note is that determination of phosphorylation stoichiometry remains as a challenge. This study first expanded a recent genetic code expansion approach to produce phosphorylated rat nNOSμ and nNOSα proteins through site-specific incorporation of phosphoserine (pSer) at residues 1446 and 1412, respectively. A quantitative mass spectrometric approach was then developed to analyze unphosphorylated peptides in phosphatase-treated and -untreated phospho-nNOS proteins. The observed pSer-incorporation efficiency consistently exceeded 80%, showing high pSer-incorporation efficiency. Notably, under l-Arginine-depleted conditions, pSer1412 nNOSα presented a significant reduction in superoxide generation, whereas pSer1446 nNOSμ exhibited the opposite effect, compared to their unphosphorylated counterparts. This suggests that phosphorylation at the C-terminal tail has a regulatory effect on NOS activity that may differ between variants and isoforms. Furthermore, the methodologies for incorporating pSer into large protein and quantifying the percentage of pSer should be applicable to other protein systems.

Postcontractile blood oxygenation level-dependent (BOLD) response in Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is characterized by a progressive replacement of muscle by fat and fibrous tissue, muscle weakness, and loss of functional abilities. Impaired vasodilatory and blood flow responses to muscle activation have also been observed in DMD and associated with mislocalization of neuronal nitric oxide synthase mu (nNOSμ) from the sarcolemma. The objective of this study was to determine whether the postcontractile blood oxygen level-dependent (BOLD) MRI response is impaired in DMD and correlated with established markers of disease severity in DMD, including MRI muscle fat fraction (FF) and clinical functional measures. Young boys with DMD (n = 16, 5-14 yr) and unaffected controls (n = 16, 5-14 yr) were evaluated using postcontractile BOLD, FF, and functional assessments. The BOLD response was measured following five brief (2 s) maximal voluntary dorsiflexion contractions, each separated by 1 min of rest. FFs from the anterior compartment lower leg muscles were quantified via chemical shift-encoded imaging. Functional abilities were assessed using the 10 m walk/run and the 6-min walk distance (6MWD). The peak BOLD responses in the tibialis anterior and extensor digitorum longus were reduced (P < 0.001) in DMD compared with controls. Furthermore, the anterior compartment peak BOLD response correlated with function (6MWD ρ = 0.87, P < 0.0001; 10 m walk/run time ρ = -0.78, P < 0.001) and FF (ρ = -0.52, P = 0.05). The reduced postcontractile BOLD response in DMD may reflect impaired microvascular function. The relationship observed between the postcontractile peak BOLD response and functional measures and FF suggests that the BOLD response is altered with disease severity in DMD.NEW & NOTEWORTHY This study examined the postcontractile blood oxygen level-dependent (BOLD) response in boys with Duchenne muscular dystrophy (DMD) and unaffected controls, and correlated this measure to markers of disease severity. Our findings indicate that the postcontractile BOLD response is impaired in DMD after brief muscle contractions, is correlated to disease severity, and may be valuable to implement in future studies to evaluate treatments targeting microvascular function in DMD.

Effects of PDE5 inhibition on dystrophic muscle following an acute bout of downhill running and endurance training.

Lack of sarcolemma-localized neuronal nitric oxide synthase mu (nNOSμ) contributes to muscle damage and fatigue in dystrophic muscle. In this study, we examined the effects of compensating for lack of nNOSμ with a phosphodiesterase type 5 (PDE5) inhibitor in mdx mice following downhill running and endurance training. Dystrophic mice (mdx) were treated with sildenafil citrate and compared with untreated mdx and wild-type mice after an acute bout of downhill running and during a progressive low-intensity treadmill running program (5 days/wk, 4 wk). Magnetic resonance imaging (MRI) and spectroscopy (MRS) transverse relaxation time constant (T2) of hindlimb and forelimb muscles were measured as a marker of muscle damage after downhill running and throughout training. The MRI blood oxygenation level dependence (BOLD) response and 31phosphorus MRS (31P-MRS) data were acquired after stimulated muscle contractions. After downhill running, the increase in T2 was attenuated (P < 0.05) in treated mdx and wild-type mice compared with untreated mdx. During training, resting T2 values did not change in wild-type and mdx mice from baseline values; however, the running distance completed during training was greater (P < 0.05) in treated mdx (>90% of target distance) and wild-type (100%) than untreated mdx (60%). The post-contractile BOLD response was greater (P < 0.05) in treated mdx that trained than untreated mdx, with no differences in muscle oxidative capacity, as measured by 31P-MRS. Our findings indicate that PDE5 inhibition reduces muscle damage after a single bout of downhill running and improves performance during endurance training in dystrophic mice, possibly because of enhanced microvascular function. NEW & NOTEWORTHY This study examined the combined effects of PDE5 inhibition and exercise in dystrophic muscle using high-resolution magnetic resonance imaging and spectroscopy. Our findings demonstrated that sildenafil citrate reduces muscle damage after a single bout of downhill running, improves endurance-training performance, and enhances microvascular function in dystrophic muscle. Collectively, the results support the combination of exercise and PDE5 inhibition as a therapeutic approach in muscular dystrophies lacking nNOSμ.

Normal increases in insulin-stimulated glucose uptake after ex vivo contraction in neuronal nitric oxide synthase mu (nNOSμ) knockout mice.

Nitric oxide (NO) is involved in skeletal muscle glucose uptake during exercise and also in the increase in insulin sensitivity after exercise. Given that neuronal nitric oxide synthase (NOS) isoform mu (nNOSμ) is a major isoform of NOS in skeletal muscle, we examined if the increase in skeletal muscle insulin-stimulated glucose uptake 3.5h following ex vivo contraction of extensor digitorum longus (EDL) is reduced in muscles from nNOSμ+/- and nNOSμ-/- mice compared with nNOSμ+/+ mice. 3.5h post-contraction/basal, muscles were exposed to saline or insulin (120μU/ml) with or without the presence of the NOS inhibitor NG-monomethyl-L-arginine (L-NMMA) during the last 30min and glucose uptake was determined by radioactive tracers. Skeletal muscle insulin-stimulated glucose uptake from nNOSμ+/+, nNOSμ+/-, and nNOSμ-/- mice increased approximately twofold 3.5h following ex vivo contraction when compared to rest. L-NMMA significantly attenuated this increase in muscle insulin-stimulated glucose uptake by around 50%, irrespective of genotype. Low levels of NOS activity were detected in muscles from nNOSμ-/- mice. In conclusion, NO mediates increases in mouse skeletal muscle insulin response following ex vivo contraction independently of nNOSμ.

Perspective: Spectrin-Like Repeats in Dystrophin Have Unique Binding Preferences for Syntrophin Adaptors That Explain the Mystery of How nNOSμ Localizes to the Sarcolemma.

Dystrophin is a massive multi-domain protein composed of specialized amino and carboxyl termini that are separated by 24 spectrin-like repeats. Dystrophin performs critical structural and signaling roles that are indispensable for the functional integrity of skeletal muscle. Indeed, the loss of dystrophin protein expression causes the muscle wasting disease, Duchenne muscular dystrophy (DMD). Substantial progress has been made in defining the functions of the domains of dystrophin, which has proven invaluable for the development of miniaturized dystrophin gene and exon skipping therapies for DMD. However, a long-standing mystery regarding dystrophin function is how dystrophin, and its adaptor and neuronal nitric oxide synthase mu (nNOSμ) binding partner α-syntrophin, cooperate to localize nNOSμ to the sarcolemma. Only when localized to the sarcolemma can nNOSμ override sympathetic vasoconstriction and prevent functional ischemia in contracting muscles. Current evidence suggests that spectrin-like repeat 17 of dystrophin and α-syntrophin cooperate to localize nNOSμ to the sarcolemma. However, the exact mechanism remains unclear and controversial because of equivocal evidence for direct binding of dystrophin and nNOSμ. Recently, an important study identified a novel α-syntrophin binding site within spectrin-like repeat 17, leading to a new model whereby α-syntrophin recruits nNOSμ to the sarcolemmal dystrophin complex by binding spectrin-like repeat 17. This model finally appears to solve the mystery of the dual requirement for dystrophin and α-syntrophin for sarcolemmal nNOSμ localization. The aim of the current perspective is to highlight this major advance in understanding of dystrophin’s role in localizing nNOSμ and its implications for current trials.

Dystrophin Threshold Level Necessary for Normalization of Neuronal Nitric Oxide Synthase, Inducible Nitric Oxide Synthase, and Ryanodine Receptor-Calcium Release Channel Type 1 Nitrosylation in Golden Retriever Muscular Dystrophy Dystrophinopathy.

At present, the clinically most advanced strategy to treat Duchenne muscular dystrophy (DMD) is the exon-skipping strategy. Whereas antisense oligonucleotide-based clinical trials are underway for DMD, it is essential to determine the dystrophin restoration threshold needed to ensure improvement of muscle physiology at the molecular level. A preclinical trial has been conducted in golden retriever muscular dystrophy (GRMD) dogs treated in a forelimb by locoregional delivery of rAAV8-U7snRNA to promote exon skipping on the canine dystrophin messenger. Here, we exploited rAAV8-U7snRNA-transduced GRMD muscle samples, well characterized for their percentage of dystrophin-positive fibers, with the aim of defining the threshold of dystrophin rescue necessary for normalization of the status of neuronal nitric oxide synthase mu (nNOSμ), inducible nitric oxide synthase (iNOS), and ryanodine receptor-calcium release channel type 1 (RyR1), crucial actors for efficient contractile function. Results showed that restoration of dystrophin in 40% of muscle fibers is needed to decrease abnormal cytosolic nNOSμ expression and to reduce overexpression of iNOS, these two parameters leading to a reduction in the NO level in the muscle fibers. Furthermore, the same percentage of dystrophin-positive fibers of 40% was associated with the normalization of RyR1 nitrosylation status and with stabilization of the RyR1-calstabin1 complex that is required to facilitate coupled gating. We concluded that a minimal threshold of 40% of dystrophin-positive fibers is necessary for the reinstatement of central proteins needed for proper muscle contractile function, and thus identified a rate of dystrophin expression significantly improving, at the molecular level, the dystrophic muscle physiology.

Abstract 20426: Study of S-Nitrosylation in Duchenne Muscular Dystrophy

Introduction: Severe cardiac myopathy occurs with Duchenne muscular dystrophy (DMD), a fatal disease characterized by absence of functional dystrophin protein. The mechanisms of the disease in cardiac muscle and whether it mimics some of the pathophysiological mechanisms present in skeletal muscle are not clear. The loss of dystrophin was reported to disrupt neuronal nitric oxide synthase mu, presumably altering redox-signaling. Nitric oxide-induced S-nitrosylation (SNO) on cysteine residues is known as a redox-sensor, which responds to cellular dynamic redox-environment. Here we identified and quantified the SNO-modified sites of cardiac tissue in dystrophy model using a novel proteomics approach. Methods: SNO-proteins from cardiac tissues of 10 months-old mdx/utrn+/- (n=3) and C57BL/6 (n=3) mice were detected with our improved TMT (tandem mass tag)-switch technique coupled with liquid chromatography/tandem mass spectrometry. The identified SNO of each site was quantified across DMD and WT samples and the fold change of SNO on each site in DMD compared to WT was displayed as ‘SNO-index (I)’, calculated by SNO DMD /SNO WT . Results: Proteomics analysis identified total 866 SNO-modified peptides on 287 SNO-proteins in the dystrophy cardiac tissue. The magnitudes of SNO-fold change were various across peptides (I=0.1 least to 1702.7 most). 32% of these, 278 peptides were more than two-fold oxidized in DMD than WT. Interestingly, a termed ‘Dilated cardiomyopathy’ protein group by KEGG-pathway analysis has extremely oxidized sites than other groups, which are more than 10-folds S-nitrosylated in DMD. Myosin-6 at Cys949 and Cys1750 (I=11.4, 117.3), Cys432 of myosin-binding protein C (I=135.0) and Cys191 of myosin light chain 3 (I=81.2) displayed a considerable SNO-increase in dystrophy sample and Cys35 of troponin C was also more modified (I=2.9), while other sites such as Cys397 of creatine kinase S-type was less oxidized (I=0.26) in DMD. Conclusions: Our novel TMT-switch assay allowed quantification of the very labile SNO-modification as well as site-identification. More than 500 cysteine residues sensed the oxidative stimuli and each had a specific susceptibility to SNO. The SNO-based redox switches were shifted in DMD compared with WT.

Differential calmodulin-modulatory and electron transfer properties of neuronal nitric oxide synthase mu compared to the alpha variant

Neuronal nitric oxide synthase μ (nNOSμ) contains 34 additional residues in an autoregulatory element compared to nNOSα. Cytochrome c and flavin reductions in the absence of calmodulin (CaM) were faster in nNOSμ than nNOSα, while rates in the presence of CaM were smaller. The magnitude of stimulation by CaM is thus notably lower in nNOSμ. No difference in NO production was observed, while electron transfer between the FMN and heme moieties and formation of an inhibitory ferrous-nitrosyl complex were slower in nNOSμ. Thus, the insert affects electron transfer rates, modulation of electron flow by CaM, and heme–nitrosyl complex formation.

Functional Deficits in nNOSμ-Deficient Skeletal Muscle: Myopathy in nNOS Knockout Mice

Skeletal muscle nNOSμ (neuronal nitric oxide synthase mu) localizes to the sarcolemma through interaction with the dystrophin-associated glycoprotein (DAG) complex, where it synthesizes nitric oxide (NO). Disruption of the DAG complex occurs in dystrophinopathies and sarcoglycanopathies, two genetically distinct classes of muscular dystrophy characterized by progressive loss of muscle mass, muscle weakness and increased fatigability. DAG complex instability leads to mislocalization and downregulation of nNOSμ; but this is thought to play a minor role in disease pathogenesis. This view persists without knowledge of the role of nNOS in skeletal muscle contractile function in vivo and has influenced gene therapy approaches to dystrophinopathy, the majority of which do not restore sarcolemmal nNOSμ. We address this knowledge gap by evaluating skeletal muscle function in nNOS knockout (KN1) mice using an in situ approach, in which the muscle is maintained in its normal physiological environment. nNOS-deficiency caused reductions in skeletal muscle bulk and maximum tetanic force production in male mice only. Furthermore, nNOS-deficient muscles from both male and female mice exhibited increased susceptibility to contraction-induced fatigue. These data suggest that aberrant nNOSμ signaling can negatively impact three important clinical features of dystrophinopathies and sarcoglycanopathies: maintenance of muscle bulk, force generation and fatigability. Our study suggests that restoration of sarcolemmal nNOSμ expression in dystrophic muscles may be more important than previously appreciated and that it should be a feature of any fully effective gene therapy-based intervention.

A Novel nNOS Signaling Pathway Regulates Skeletal Muscle Size, Strength and Fatigue Resistance

Nitric oxide (NO) is a highly versatile signaling molecule which participates in a broad array of pathways in the cardiovascular, immune and nervous systems. We present further evidence for this physiological diversity by identifying a novel nNOS signaling pathway in skeletal muscle. NO is synthesized by neuronal nitric oxide synthase mu isoenzyme (nNOSμ) in skeletal muscle. nNOSμ is found associated with the dystrophin‐associated glycoprotein complex and in a soluble pool. During investigation of the soluble nNOS pool, we identified a splice variant of nNOS called nNOSβ localized to the Golgi complex. Soluble guanylyl cyclase and protein kinase G also localized to the Golgi suggesting that a classical NO–cGMP signaling pathway functioned at the Golgi complex. Loss of nNOSβ activity resulted in reduced muscle mass with muscles exhibiting lower force‐generating capacity, even after correcting for decreased muscle size. Furthermore, the absence of nNOS decreased the ability of muscle to resist exercise‐induced fatigue. In summary, we have identified a novel nNOS‐signaling pathway that regulates skeletal muscle size and strength and provided the first evidence that differential targeting of nNOS isozymes facilitates the creation of spatially and functionally distinct nNOS signaling pathways by localized synthesis of an otherwise directionless free radical gas. Furthermore, these results make nNOSβ of potential relevance to myopathies and dystrophies where preventing the loss of muscle bulk and strength is of clinical importance.

AMPK signaling in contracting human skeletal muscle: acetyl-CoA carboxylase and NO synthase phosphorylation.

AMP-activated protein kinase (AMPK) is a metabolic stress-sensing protein kinase responsible for coordinating metabolism and energy demand. In rodents, exercise accelerates fatty acid metabolism, enhances glucose uptake, and stimulates nitric oxide (NO) production in skeletal muscle. AMPK phosphorylates and inhibits acetyl-coenzyme A (CoA) carboxylase (ACC) and enhances GLUT-4 translocation. It has been reported that human skeletal muscle malonyl-CoA levels do not change in response to exercise, suggesting that other mechanisms besides inhibition of ACC may be operating to accelerate fatty acid oxidation. Here, we show that a 30-s bicycle sprint exercise increases the activity of the human skeletal muscle AMPK-alpha1 and -alpha2 isoforms approximately two- to threefold and the phosphorylation of ACC at Ser(79) (AMPK phosphorylation site) approximately 8.5-fold. Under these conditions, there is also an approximately 5.5-fold increase in phosphorylation of neuronal NO synthase-mu (nNOSmu;) at Ser(1451). These observations support the concept that inhibition of ACC is an important component in stimulating fatty acid oxidation in response to exercise and that there is coordinated regulation of nNOSmu to protect the muscle from ischemia/metabolic stress.