Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy
Yi Lai, … , Ronald L. Terjung, Dongsheng Duan
Yi Lai, … , Ronald L. Terjung, Dongsheng Duan
Published March 2, 2009; First published February 23, 2009
Citation Information: J Clin Invest. 2009;119(3):624-635. https://doi.org/10.1172/JCI36612.
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Research Article

Dystrophins carrying spectrin-like repeats 16 and 17 anchor nNOS to the sarcolemma and enhance exercise performance in a mouse model of muscular dystrophy

Abstract

Sarcolemma-associated neuronal NOS (nNOS) plays a critical role in normal muscle physiology. In Duchenne muscular dystrophy (DMD), the loss of sarcolemmal nNOS leads to functional ischemia and muscle damage; however, the mechanism of nNOS subcellular localization remains incompletely understood. According to the prevailing model, nNOS is recruited to the sarcolemma by syntrophin, and in DMD this localization is altered. Intriguingly, the presence of syntrophin on the membrane does not always restore sarcolemmal nNOS. Thus, we wished to determine whether dystrophin functions in subcellular localization of nNOS and which regions may be necessary. Using in vivo transfection of dystrophin deletion constructs, we show that sarcolemmal targeting of nNOS was dependent on the spectrin-like repeats 16 and 17 (R16/17) within the rod domain. Treatment of mdx mice (a DMD model) with R16/17-containing synthetic dystrophin genes effectively ameliorated histological muscle pathology and improved muscle strength as well as exercise performance. Furthermore, sarcolemma-targeted nNOS attenuated α-adrenergic vasoconstriction in contracting muscle and improved muscle perfusion during exercise as measured by Doppler and microsphere circulation. In summary, we have identified the dystrophin spectrin-like repeats 16 and 17 as a novel scaffold for nNOS sarcolemmal targeting. These data suggest that muscular dystrophy gene therapies based on R16/17-containing dystrophins may yield better clinical outcomes than the current therapies.

Authors

Yi Lai, Gail D. Thomas, Yongping Yue, Hsiao T. Yang, Dejia Li, Chun Long, Luke Judge, Brian Bostick, Jeffrey S. Chamberlain, Ronald L. Terjung, Dongsheng Duan

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Figure 1

Sarcolemmal nNOS localization depends on the rod, but not the C-terminal, domain of dystrophin.

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Sarcolemmal nNOS localization depends on the rod, but not the C-terminal...
(A) Schematic outline of the full-length and synthetic dystrophin constructs. “Yes” indicates that nNOS is recruited to the sarcolemma by the construct; “No” indicates that the construct cannot restore nNOS to the sarcolemma. Dotted boxes denote deleted regions. (B) The DH2–R15 minidystrophin anchors nNOS to the sarcolemma. Representative photomicrographs of dystrophin immunostaining, nNOS activity staining, and nNOS immunostaining in the DH2–R15 minigene plasmid–transfected mdx muscle. Dystrophin was revealed by epitope-specific antibodies (H1, R11, R16, R17, R18, and H3). The H1 antibody only recognizes human dystrophin (Hum Dys). Other dystrophin antibodies recognize both human and mouse dystrophin. Asterisks indicate a minigene-transfected myofiber; arrows, revertant myofibers. Scale bar: 20 μm. (C) Sarcolemmal nNOS localization does not require the C-terminal domain, nor is it dependent on the repeats adjacent to R16/17. Representative photomicrographs of human dystrophin/nNOS double immunostaining and nNOS activity staining on serial sections from the DH2–R15/DR18–19, DH2–R15/DC, and DR3–15/DR18–23/DC plasmid–transfected mdx muscles. Asterisks indicate dystrophin plasmid–transfected fibers; crosses, revertant myofibers. Scale bar: 50 μm.