Mini-dystrophin gene transfer in mdx4cv diaphragm muscle fibers increases sarcolemmal stability.

To date, all dystrophin gene transfer studies have been performed on mdx hindlimb skeletal muscles which in comparison to the severe deficits seen in muscles from patients afflicted with Duchenne muscular dystrophy (DMD), exhibit only modest morphological and functional changes. Since the mdx diaphragm muscle presents the same pathophysiological alterations characteristic of DMD muscles, we therefore injected recombinant plasmid DNA encoding the dystrophin mini-gene (pRSVdy-B) into diaphragm muscles of 10-week-old mdx4cv mice and examined the physiological consequences of dystrophin expression in a muscle that has undergone a phase of massive degeneration and regeneration. Immunoperoxidase and immunofluorescence experiments revealed that 1 and 3 weeks following gene transfer, approximately 17% of the fibers in a bundle of diaphragm muscle expressed dystrophin at the sarcolemma. Most importantly, this level of dystrophin expression was sufficient to protect all fibers present within these diaphragm muscle bundles from the damaging effects of repetitive lengthening contractions. In addition, dystrophin expression partially restored the ability of transduced mdx4cv muscle bundles to generate isometric tetanic tension following lengthening contractions. These results show that mini-dystrophin expression leads to rapid and significant functional improvements in diaphragm muscles of mdx4cv mice. Although these data provide encouraging results for future therapeutic strategies aimed at curing DMD, additional work will none the less be necessary to determine the full impact of dystrophin gene replacement. In this context, it is clear from the data presented here that the diaphragm muscle of the mdx mouse is an invaluable model system to address this critical issue.