α1-Syntrophin Gene Disruption Results in the Absence of Neuronal-type Nitric-oxide Synthase at the Sarcolemma but Does Not Induce Muscle Degeneration

Kameya, Shuhei; Miyagoe, Yuko; Nonaka, Ikuya; Ikemoto, Takaaki; Endo, Makoto; Hanaoka, Kazunori; Nabeshima, Yo‐ichi; Takeda, Shin‐ichi

doi:10.1074/jbc.274.4.2193

Cited by 165 publications

(176 citation statements)

References 30 publications

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“…It was reported that nNOS is anchored at the subsarcolemma through its binding to the rod domain of dystrophin at the 16 and 17 rod repeats encoded by exons 42-45 and by interaction with α1-syntrophin (20,21). The truncated dystrophin induced by skipping exons 45-55 would lack half of the 17 rod repeat, thus disturbing the site-responsible anchoring of nNOS and possibly rendering its subsarcolemmal localization unstable.…”

Section: Discussionmentioning

confidence: 99%

Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery

Aoki

Yokota

Nagata

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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Duchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is caused by lack of dystrophin. One of the most promising therapeutic approaches is antisense-mediated elimination of frame-disrupting mutations by exon skipping. However, this approach faces two major hurdles: limited applicability of each individual target exon and uncertain function and stability of each resulting truncated dystrophin. Skipping of exons 45-55 at the mutation hotspot of the DMD gene would address both issues. Theoretically it could rescue more than 60% of patients with deletion mutations. Moreover, spontaneous deletions of this specific region are associated with asymptomatic or exceptionally mild phenotypes. However, such multiple exon skipping of exons 45-55 has proved technically challenging. We have therefore designed antisense oligo (AO) morpholino mixtures to minimize self-or heteroduplex formation. These were tested as conjugates with cell-penetrating moieties (vivo-morpholinos). We have tested the feasibility of skipping exons 45-55 in H2K-mdx52 myotubes and in mdx52 mice, which lack exon 52. Encouragingly, with mixtures of 10 AOs, we demonstrated skipping of all 10 exons in vitro, in H2K-mdx52 myotubes and on intramuscular injection into mdx52 mice. Moreover, in mdx52 mice in vivo, systemic injections of 10 AOs induced extensive dystrophin expression at the subsarcolemma in skeletal muscles throughout the body, producing up to 15% of wild-type dystrophin protein levels, accompanied by improved muscle strength and histopathology without any detectable toxicity. This is a unique successful demonstration of effective rescue by exon 45-55 skipping in a dystrophin-deficient animal model. personalized medicine | nucleic acid therapy | molecular therapy | oligonucleotides | gene therapy D uchenne muscular dystrophy (DMD), the commonest form of muscular dystrophy, is characterized by progressive deterioration of muscle function (1). DMD is caused mainly by frame-shifting deletion or nonsense mutations in the DMD gene, which encodes the protein dystrophin (2). At the milder end of the disease spectrum, Becker muscular dystrophy (BMD) is a form of dystrophin deficiency that presents with a large spectrum of severities, from borderline DMD to almost asymptomatic cases. BMD typically results from in-frame deletions in the DMD gene that allow the expression of limited amounts of an internally truncated but partly functional protein (3).Skipping of exons in DMD muscle so as to restore an in-frame and asymptomatic or very mild Becker-like transcript is among the more promising therapeutic approaches to treatment of DMD (4). To this end, systemic administration of antisense oligonucleotides (AOs) targeting specific exon(s) in the DMD gene has been shown to restore the reading frame and induce body-wide production of partially functional BMD-like dystrophin in mouse and dog models of DMD (5-7). Recently, phase I/II human clinical trials with AOs targeting exon 51 have been completed (8, 9).Although promising, future developm...

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Section: Discussionmentioning

confidence: 99%

Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery

Aoki

Yokota

Nagata

et al. 2012

Proc. Natl. Acad. Sci. U.S.A.

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141

124

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“…Fluorescent images were viewed with an AxioCam HRc microscope from Carl Zeiss Vision GmbH (Munich, Germany) and recorded with a digital camera using Axiovision 4 image acquisition software. Haematoxylin and eosin (H&E) staining of tissue sections, labelling of nuclei with DAPI, and histochemical staining for succinate dehydrogenase and NADPH diaphorase were performed by standard procedures (Kameya et al, 1999;Yasuda et al, 2002;Dowling et al, 2004).…”

Section: Immunofluorescence Microscopy and Histochemical Stainingmentioning

confidence: 99%

Expression of the skeletal muscle dystrophin–dystroglycan complex and syntrophin-nitric oxide synthase complex is severely affected in the type 2 diabetic Goto–Kakizaki rat

Mulvey

Harno

Keenan

et al. 2005

European Journal of Cell Biology

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The inability of insulin to stimulate glucose metabolism in skeletal muscle fibres is a classic characteristic of type 2 diabetes. Using the non-obese Goto-Kakizaki rat as an established animal model of this type of diabetes, sucrose gradient centrifugation studies were performed and confirmed the abnormal subcellular location of the glucose transporter GLUT4. In addition, this analysis revealed an unexpected drastic reduction in the surface membrane marker b-dystroglycan, a dystrophin-associated glycoprotein. Based on this finding, a comprehensive immunoblotting survey was conducted which showed a dramatic decrease in the Dp427 isoform of dystrophin and the a/b-dystroglycan subcomplex, but not in laminin, sarcoglycans, dystrobrevin, and excitation-contraction-relaxation cycle elements. Thus, the backbone of the trans-sarcolemmal linkage between the extracellular matrix and the actin membrane cytoskeleton might be structurally impaired in diabetic fibres. Immunohistochemical studies revealed that the reduction in the dystrophin-dystroglycan complex does not induce obvious signs of muscle pathology, and is neither universal in all fibres, nor fibre-type specific. Most importantly, the expression of a-syntrophin and the syntrophinassociated neuronal isoform of nitric oxide synthase, nNOS, was demonstrated to be severely reduced in diabetic fibres. The loss of the dystrophin-dystroglycan complex and the syntrophin-nNOS complex in selected fibres suggests a weakening of the sarcolemma, abnormal signalling and probably a decreased cytoprotective mechanism in diabetes. Impaired anchoring of the cortical actin cytoskeleton via dystrophin might interfere with the proper recruitment of the glucose transporter to the surface membrane, following stimulation by insulin or muscle contraction. This may, at least partially, be responsible for the insulin resistance in diabetic skeletal muscles.

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“…The sarcolemmal nNOSμ association requires α-syntrophin, dystrophin, and α-dystrobrevin (Brenman et al 1995(Brenman et al , 1996Grady et al 2000;Adams et al 2000Adams et al , 2008. In mice, nNOSμ is not only localized at the sarcolemma, but is also normally expressed at relatively high levels in the cytoplasm (Chang et al 1996;Kameya et al 1999;Thomas et al 2003). However, in human muscle approximately 75% of nNOSμ is associated with the sarcolemmal DGC, suggesting species-specific differences in nNOSμ distribution (Chang et al 1996).…”

Section: Introductionmentioning

confidence: 99%

nNOS regulation of skeletal muscle fatigue and exercise performance

Percival

2011

Biophys Rev

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Neuronal nitric oxide synthases (nNOS) are Ca 2+ /calmodulin-activated enzymes that synthesize the gaseous messenger nitric oxide (NO). nNOSμ and the recently described nNOSβ, both spliced nNOS isoforms, are important enzymatic sources of NO in skeletal muscle, a tissue long considered to be a paradigmatic system for studying NO-dependent redox signaling. nNOS is indispensable for skeletal muscle integrity and contractile performance, and deregulation of nNOSμ signaling is a common pathogenic feature of many neuromuscular diseases. Recent evidence suggests that both nNOSμ and nNOSβ regulate skeletal muscle size, strength, and fatigue resistance, making them important players in exercise performance. nNOSμ acts as an activity sensor and appears to assist skeletal muscle adaptation to new functional demands, particularly those of endurance exercise. Prolonged inactivity leads to nNOS-mediated muscle atrophy through a FoxO-dependent pathway. nNOS also plays a role in modulating exercise performance in neuromuscular disease. In the mdx mouse model of Duchenne muscular dystrophy, defective nNOS signaling is thought to restrict contractile capacity of working muscle in two ways: loss of sarcolemmal nNOSμ causes excessive ischemic damage while residual cytosolic nNOSμ contributes to hypernitrosylation of the ryanodine receptor, causing pathogenic Ca 2+ leak. This defect in Ca 2+ handling promotes muscle damage, weakness, and fatigue. This review addresses these recent advances in the understanding of nNOS-dependent redox regulation of skeletal muscle function and exercise performance under physiological and neuromuscular disease conditions.

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α1-Syntrophin Gene Disruption Results in the Absence of Neuronal-type Nitric-oxide Synthase at the Sarcolemma but Does Not Induce Muscle Degeneration

Cited by 165 publications

References 30 publications

Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery

Bodywide skipping of exons 45–55 in dystrophic mdx52 mice by systemic antisense delivery

Expression of the skeletal muscle dystrophin–dystroglycan complex and syntrophin-nitric oxide synthase complex is severely affected in the type 2 diabetic Goto–Kakizaki rat

nNOS regulation of skeletal muscle fatigue and exercise performance

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