Molecular Genetics and Metabolism 82 (2004) 312–320 www.elsevier.com/locate/ymgme 1096-7192/$ - see front matter  2004 Elsevier Inc. All rights reserved. doi:10.1016/j.ymgme.2004.06.006 Expression of a NOS transgene in dystrophin-deWcient muscle reduces muscle membrane damage without increasing the expression of membrane-associated cytoskeletal proteins James G. Tidball a,b,¤ and Michelle Wehling-Henricks a a Department of Physiological Science, University of California, Los Angeles, CA 90095, USA b Department of Pathology and Laboratory Medicine, University of California, Los Angeles, CA 90095, USA Received 26 March 2004; received in revised form 9 June 2004; accepted 10 June 2004 Available online 15 July 2004 Abstract Muscular dystrophy that is caused by mutation of the membrane-associated, cytoskeletal protein called dystrophin, is accompa- nied by loss of a dystrophin-associated protein complex (DPC) that includes neuronal nitric oxide synthase (nNOS). Previous work showed that expression of a nNOS transgene in the dystrophin-deWcient, mdx mouse greatly reduces muscle membrane damage. In this investigation, we test whether expression of a nNOS transgene in wild-type or mdx muscle increases expression of DPC proteins, or functionally related proteins in the integrin complex that are upregulated in dystrophin-deWciency, or aVects expression of the dys- trophin homolog, utrophin. Many members of the DPC are enriched in Western blots of cell membranes isolated from NOS trans- genic muscle, compared to wild-type. Similarly, 7-integrin and the associated cytoskeletal proteins talin and vinculin are increased in NOS transgenic, non-dystrophic muscle. However, utrophin expression is unaVected by elevated NOS expression in healthy mus- cle. A similar trend in mRNA levels for these proteins was observed by expression proWling. Analysis of membrane preparations from mdx mice and NOS transgenic mdx mice shows that expression of the NOS transgene causes signiWcant reductions in utrophin, talin, and vinculin. Expression proWling of mRNA from mdx and NOS transgenic mdx muscles also shows reduced expression of talin. Immunohistochemistry of mdx and NOS transgenic mdx muscle indicates that reduction in utrophin in NOS transgenic mdx muscle results from a decrease in regenerative Wbers that express high levels of utrophin. Together, these Wndings indicate that the NOS transgene does not reduce dystrophinopathy by increasing the expression of compensatory, structural proteins.  2004 Elsevier Inc. All rights reserved. Keywords: Nitric oxide synthase; Muscular dystrophy; Muscle pathology; Cytoskeletal proteins Introduction Null mutation of the membrane-associated, cytoskel- etal protein dystrophin results in muscle pathology that occurs in Duchenne muscular dystrophy (DMD) and in mdx mice [1]. Dystrophin-deWciency results in a mechan- ically weaker cell membrane, which is expected to be an important functional defect contributing to the disease [2,3]. In both DMD and mdx pathologies, muscles expe- rience necrosis and inXammation [4]. However, in DMD, muscle necrosis and wasting are progressive and result in early death, while in mdx mice there is a peak of necrosis and inXammation at 4 weeks of age, followed by regen- eration and a nearly normal lifespan. DiVerences in the progression of mdx and DMD pathologies have been largely attributed to increases in expression of structural proteins that can compensate for the loss of dystrophin in mdx muscles, but not in DMD muscles. This view was initially founded on the discovery that utrophin, a homolog of dystrophin, was expressed at higher levels at the membranes of regenera- tive mdx muscles, where they could possibly strengthen the weak, dystrophin-deWcient membrane [5]. However, loss of dystrophin leads to a secondary loss of the entire ¤ Corresponding author. Fax: 1-310-825-8489. E-mail address: jtidball@physci.ucla.edu (J.G. Tidball).