Sarcolemmal nNOS anchoring reveals a qualitative difference between dystrophin and utrophin

Abstract

Duchenne muscular dystrophy (DMD) is a lethal muscle disease caused by dystrophin deficiency. In normal muscle, dystrophin helps maintain sarcolemmal stability. Dystrophin also recruits neuronal nitric oxide synthase (nNOS) to the sarcolemma. Failure to anchor nNOS to the membrane leads to functional ischemia and aggravates muscle disease in DMD. Over the past two decades, a great variety of therapeutic modalities have been explored to treat DMD. A particularly attractive approach is to increase utrophin expression. Utrophin shares considerable sequence, structural and functional similarity with dystrophin. Here, we test the hypothesis that utrophin also brings nNOS to the sarcolemma. Full-length utrophin cDNA was expressed in dystrophin-deficient mdx mice by gutted adenovirus or via transgenic overexpression. Subcellular nNOS localization was determined by immunofluorescence staining, in situ nNOS activity staining and microsomal preparation western blot. Despite supra-physiological utrophin expression, we did not detect nNOS at the sarcolemma. Furthermore, transgenic utrophin overexpression failed to protect mdx muscle from exercise-associated injury. Our results suggest that full-length utrophin cannot anchor nNOS to the sarcolemma. This finding might have important implications for the development of utrophin-based DMD therapies.

Fig. 1.

Adenovirus-mediated full-length utrophin expression does not restore sarcolemmal nNOS. (A) Representative dystrophin and nNOS immunofluorescence staining photomicrographs from normal and mdx muscles. Some reverent fibers can restore nNOS (arrowhead) whereas others cannot (arrow). (B) Representative immunofluorescence staining photomicrographs at 2 months after mdx muscles were infected with gutted adenoviruses. Left panels are dystrophin and nNOS staining following human full-length dystrophin gutted adenovirus infection. Right panels are utrophin and nNOS staining following mouse full-length utrophin gutted adenovirus infection. gAd, gutted adenovirus; HDys, human dystrophin; Utr, utrophin. (C) Two examples of serial sections from full-length utrophin gutted adenovirus infected muscles stained with antibodies against utrophin (left panels) and α-syntrophin (right panels). Myofibers expressing high levels of utrophin at the sarcolemma also expressed α-syntrophin. Asterisks indicate the same myofiber in serial sections.

Fig. 2.

In situ evaluation of muscle histology and dystrophin, utrophin, syntrophin and nNOS expression. Serial muscle sections from normal BL10 (A), mdx (B), utrophin knockout (C), u-dko (D) and Fiona strain of utrophin transgenic mdx mice (E) were stained for general histology with hematoxylin and eosin (HE), and for dystrophin, utrophin, syntrophin, nNOS and nNOS activity. Representative photomicrographs from each mouse model are presented. nNOS (pAb), immunofluorescence staining for nNOS; Asterisks indicate the same myofiber in serial sections; empty arrowheads in C and E indicate neuromuscular junctions; arrows in D show damaged myofibers with infiltrated mouse immunoglobulin; filled arrowheads in E indicate myofibers with centrally located nucleus.

Fig. 3.

Western blot analysis of nNOS expression in whole-muscle lysate and microsomal preparation. (A) Representative western blot of whole-muscle lysate. (B) Representative microsomal western blot results. Rapid blue staining of a duplicated gel was included as the loading control for microsomal preparation western blot.

Fig. 4.

Continuous treadmill running leads to aggravated degeneration-regeneration and specific force reduction in the extensor digitorum longus muscle of Fiona mice. Experimental mice were divided into running and no-running groups. In the running group, mice were challenged with treadmill exercise for 8 weeks. (A) Representative photomicrographs of HE-stained muscle cross-sections from exercised mice. Arrows indicate areas of ischemic damage. (B) Specific muscle force (mean ± s.e.m., n=6 per group). Asterisk indicates a significantly lower value compared with other groups.