Abstracts from 43rd Annual Meeting The American Society for Cell Biology 

California,  13-17 december, 2003

Negamycin restores dystrophin expression in skeletal and cardiac muscles of mdx mice
M. SHIOZUKA,¹ M. ARAKAWA,¹ Y. NAKAYAMA,² T. HARA,² M. HAMADA,³ D. IKEDA,³ Y. TAKAHASHI,³ R. SAWA,³ Y. NONOMURA,³ K. SHEYKHOLESLAMI,⁴ K. KONDO,⁴ K. KAGA,⁴ S. TAKEDA,⁵ R. MATSUDA¹ ; ¹ Dept. of Life Sciences, The University of Tokyo, Tokyo, Japan, ² Dept. of Tumor Biochemistry, Tokyo Metropolitan Institute of Medical Sciences, Tokyo, Japan, ³ The Institute of Microbial Chemistry, Tokyo, Japan, ⁴ Dept. of Otolaryngology, The University of Tokyo, Tokyo, Japan, ⁵ Dept. of Molecular Therapy, National Institute of Neuroscience, Tokyo, Japan

The ability of aminoglycoside antibiotics to promote read-through of nonsense mutations has attracted interests in these drugs as potential therapeutic agents in genetic diseases. However, strong toxicity of aminoglycoside antibiotics may cause severe side effects during long-term treatment. In this study, we report that negamycin, a dipeptide antibiotic, also restores dystrophin expression in skeletal and cardiac muscles of mdx mouse; an animal model of Duchenne muscular dystrophy (DMD) with a nonsense mutation in dystrophin gene, and in cultured mdx myotubes. Dystrophin expression was confirmed by immunohistochemistry and immunoblotting. We also compared the toxicity of negamycin and gentamicin, and found negamycin to be less toxic. Furthermore, we demonstrated that negamycin bound to a partial sequence decording the eukaryotic rRNA A-site. We conclude that negamycin is a promising new candidate for chemotherapy for DMD and other genetic diseases caused by nonsense mutations.

 Calcineurin-Induced Upregulation of Utrophin Attenuates the Dystrophic Pathology in mdx Mouse Muscle
J. V. Chakkalakal,¹ M. Harrison,² E. R. Chin,³ R. N. Michel,² B. J. Jasmin¹ ; ¹ Cellular & Molecular Medicine, University of Ottawa, Ottawa, ON, Canada, ² Neuromuscular Lab, Laurentian University, Sudbury, ON, Canada, ³ Cardiovascular and Metabolic Diseases, Pfizer Global Research and Development, Groton, CT

We recently showed that mice expressing a constitutively active form of calcineurin (CnA*) have elevated levels of utrophin A in their muscles (Proc. Natl. Acad. Sci. USA, 100: 7791-7796, 2003). In the present study, we crossed these transgenic animals with mdx mice to determine whether mdx/CnA* mice would be less affected by the dystrophic process. Since expression of CnA* has been shown previously to stimulate the slow/oxidative myofiber program, we examined the expression of myosin heavy chain isoforms in mdx/CnA* and mdx mice. By immunofluorecence and RT-PCR assays, we observed in mdx/CnA* a shift in myosin heavy chain profile towards a slower, more oxidative phenotype compared to mdx muscles. In addition, we determined that expression of utrophin A and its transcript were increased by ~ 2 fold in mdx/CnA* mouse muscles. Immunodetection of IgM inside myofibers, used in this case as an index of sarcolemmal disruption, showed that the number of IgM-positive fibers was significantly reduced in muscles from mdx/CnA* mice. Consistent with these findings, we also observed that muscles from mdx/CnA* mice showed less variability in fiber size and contained fewer central nuclei. Together, these results demonstrate that enhanced calcineurin activity can have important beneficial effects on the dystrophic phenotype by stimulating the expression of utrophin A. Furthermore, these findings provide specific targets for which pharmacological strategies may be designed to enhance utrophin levels in muscles from patients with Duchenne muscular dystrophy. Funded by the MDA and CIHR.