MINI-LENGTH AND FULL-LENGTH DYSTROPHIN GENE-TRANSFER INDUCES THE RECOVERY OF NITRIC-OXIDE SYNTHASE AT THE SARCOLEMMA OF MDX4(CV) SKELETAL-MUSCLE FIBERS

In normal skeletal muscle fibers, dystrophin accumulates at the cytopl asmic face of the sarcolemma where it associates with dystrophin-assoc iated proteins (DAPs). Several studies have recently shown that neuron al isoform of nitric oxide synthase (nNOS) is also located at the sarc olemma, and that this membrane localization is mediated through intera ctions of nNOS with one of the DAPS, namely alpha 1-syntrophin. Since the lack of dystrophin in muscle fibers from Duchenne muscular dystrop hy patients and mdx mice is accompanied by an absence of sarcolemmal n NOS, we examined in the present study, whether dystrophin gene replace ment would lead to the restoration of nNOS at its appropriate subcellu lar location. To this end, tibialis anterior muscles from mdx4(cv) mic e were directly injected with plasmid DNA encoding either full-length (pRSV-dys) or mini- (pRSV-dyB; lacking exons 17-48) dystrophin. For th ese experiments, we chose to study 10-week-old mdx4(cv) mice since at this developmental stage, muscles from these mice have already undergo ne several cycles of degeneration-regeneration. Immunofluorescence exp eriments performed on serial cross-sections revealed that approximatel y 50% of the dystrophin-positive fibers also exhibited significant lev els of nNOS at their sarcolemma 2 weeks following gene transfer with p RSV-dys. Similar results were obtained with pRSV-dyB indicated that ex ons 17-48 of the dystrophin gene are not essential for the correct loc alization of nNOS in skeletal muscle fibers. Taken together with the r ecent demonstration that dystrophin gene transfer leads to significant physiological benefits our results suggest that dystrophin gene thera py using full-length or truncated dystrophin, also induces a rapid rec overy of biochemical functions.