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.

Microtubule binding distinguishes dystrophin from utrophin

“…The transgenic utrophin overexpression mouse model does not restore the localization of nNOS to the sarcolemma, making this the only thus-far documented mdx phenotype not rescued in the Fiona-mdx mouse (9,34). The second possibility is that the amount of endogenous utrophin up-regulation in the mdx mouse is simply insufficient to restore microtubule lattice organization.…”

“…The dystrophin protein consists of an amino-terminal tandem calponin-homology actin binding domain, a large rod domain composed of spectrin-like repeats and flexible hinge regions, within which lie a second actin binding domain and the neuronal nitric oxide synthase (nNOS) binding domain, and cysteine-rich and carboxyl-terminal domains (3)(4)(5)(6)(7)(8)(9). Dystrophin is enriched at subsarcolemmal protein assemblies known as costameres, where it couples actin and intermediate filaments to a membrane-associated glycoprotein complex (10)(11)(12).…”

nNOS regulation of skeletal muscle fatigue and exercise performance

“…Minidystrophin cDNAs that restore sarcolemmal nNOSμ expression improved the ability of mdx muscle to oppose sympathetic vasoconstriction, improving active muscle perfusion and substantially increasing treadmill running exercise performance (Lai et al 2009). Improved muscle perfusion reduced contractioninduced ischemic muscle damage and inflammation.…”

“…Improved muscle perfusion reduced contractioninduced ischemic muscle damage and inflammation. A very similar minidystrophin cDNA lacking the nNOSμ targeting sequence did not enhance exercise performance to the same degree (Lai et al 2009). The dystrophin minigenes that restore nNOSμ and thus enhance exercise performance are the most therapeutically effective DMD gene therapy constructs to date.…”

“…The exercise intolerance shown by DMD patients appears to arise in part from vascular dysfunction, which has long been suspected to be a critical pathogenic mechanism in DMD (Asai et al 2007;Kobayashi et al 2008;Lai et al 2009;reviewed in Percival et al 2011). Sarcolemmal nNOSμ deficiency leads to unopposed α-adrenergic receptor-mediated vasoconstriction in the active hindlimbs of mice mdx and contracting forearms of DMD patients (Thomas et al 1998;Sander et al 2000).…”