Joshua T. Selsby1,2, Kevin Morine2, Klara Pendrak2, Z. Tian3, Erica Blanco3, Elisabeth R Barton3, H Lee Sweeney2. 1Animal Science, Iowa State University, Ames, IA, 2Physiology, 3Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA

Increased PGC-1α, either through a transgenic animal or gene transfer, provides therapeutic benefit to dystrophic skeletal muscle  likely by increasing slow and oxidative protein expression. Resveratrol has been shown to activate SIRT-1, a known deacetylase capable of acting on and increasing the activity of PGC-1α. The purpose of this investigation was to determine if resveratrol supplementation could rescue dystrophic skeletal muscle. We hypothesized that daily feeding of resveratrol would improve muscle function in a similar fashion as PGC-1α overexpression. To test this hypothesis, 1-mo old mdx mice were either fed a control diet (Con) or a diet containing resveratrol at 100 mg/kg/day (Res) for 8 wks. Following intervention, absolute muscle mass was reduced 18-30% in the Res group compared to Con, however, body mass was also reduced in the Res group by 20% compared to Con. Relative muscle mass was similar between groups, except for the EDL, which was significantly smaller in the Res group. Solei in the Res group were more fatigue resistant than in the Con group, however, resistance to contraction induced injury was similar between groups. These data indicate that partial rescue of dystrophic pathology is possible through resveratrol. Perhaps, alternative sources or doses of resveratrol will provide a greater response.

Joshua T. Selsby1,2, Kevin Morine2, Klara Pendrak2, Z. Tian3, Erica Blanco3, Elisabeth R Barton3, H Lee Sweeney2. 1Animal Science, Iowa State University, Ames, IA, 2Physiology, 3Anatomy and Cell Biology, University of Pennsylvania, Philadelphia, PA

PGC-1α has received a great deal of attention due to its potential to induce oxidative and slow proteins. The purpose of this investigation was to: 1) extend observations made in an initial study demonstrating transgenic over-expression of PGC-1α reduced dystrophic pathology 2) eliminate effects that could have taken place during development. Neonatal mdx mice were injected in the right hind limb with 1x1011 gc of AAV causing overexpression of PGC-1α and sacrificed at 4 (n=6) or 6 wks of age (n=5). Muscle mass was reduced in limbs over-expressing PGC- 1α at 4 and 6 wks, however, tetantic force and specific force in the soleus and EDL were either maintained or improved when compared to control limbs. PGC-1α over-expression caused EDLs to be more resistant to damage at 6 wks and the soleus to be more fatigue resistant at 4 and 6 wks compared to control limbs. PGC- 1α increased expression of slow proteins as utrophin was increased nearly 2-fold and type I myosin heavy chain nearly 3-fold as well as expression of oxidative proteins as cytochrome C and uncoupling protein-1 were increased approximately 2-fold, complex IV subunit IV (cytochrome C oxidase) was increased 1.5- fold and myoglobin was increased 3-fold in limbs over-expressing PGC-1α compared to control limbs. These data demonstrate the potential therapeutic role of the PGC-1α pathway for dystrophic skeletal muscle.

Jong-Hee Kim1, BR Macias2, C Canon2, S Courtney2, John M Lawler2. 1Health and Kinesiology, Texas A&M University, College Station, TX, 2Texas A&M University, College Station, TX Duchenne muscular dystrophy (DMD) is characterized by devastating muscle degeneration associated with oxidative stress, loss of contractile tissue, muscle atrophy, muscle weakness and increased fibrosis in respiratory and locomotor muscles. We tested the hypothesis that protection against oxidative stress via the catalase/superoxide dismutase mimetic EUK-134 will prevent reduction of diaphragm and skeletal muscle mass/body mass during the early inflammatory phase (20-28 days) in mdx mice. C57BL(wild type) and mdx mice were given EUK-134 (30mg/kg body weight/day, i.p., injection) for 8 days, beginning at 20 days of age. Body mass was significantly lower in mdx mice (-37.7%) than wild type but, EUK-134 increased body mass by 15.5% in mdx mice. Absolute muscle mass was lower in diaphragm (-44.3%), gastrocnemius (-53.6%), tibialis anterior (-58.1%) in mdx mice. Muscle mass/body mass was lower in diaphragm (-11.4%), gastrocnemius (-26.3%), tibialis anterior (-34.4%), but not heart, plantaris, soleus, extensor digitorum longus in mdx mice. EUK- 134 had a significant positive effect in protecting against reduced muscle mass/body mass in diaphragm (+38.7%) and gastrocnemius (+27.1%) in mdx mice. These data indicate that EUK-134 provide protection against reduced muscle mass/body mass in diaphragm and gastrocnemius in mdx mice during this early phase of muscular dystrophy.

Marni Della Boppart1, Stephen J Kaufman2. 1Kinesiology and Community Health, 2Cell and Developmental Biology, University of Illinois, Urbana, IL

MCK-driven transgenic expression of the alpha7 integrin can ameliorate pathology in a mouse model of Duchenne muscular dystrophy (mdx/utrn-/-) and thus compensate for the loss of dystrophin in diseased mice. In spite of the beneficial effects of the alpha7 integrin in protecting mice from dystrophy, identification of molecular signaling events responsible for these changes remain to be established. PURPOSE: To determine a role for signaling in the amelioration of muscular dystrophy by alpha7 integrin. METHODS: Five wk wild type, mdx/utrn-/-, and alpha7BX2-mdx/utrn-/- gastrocnemius muscles (n=3-8/group) were dissected and extracted. Activation of PI3K, ILK, AKT, mTOR, p70S6K, GSK-3, and p38 was measured using in vitro activity assays or phosphospecific antibodies and western blotting. RESULTS: Significant increases in ILK activity (2.0-fold), AKT- (P) (2.3-fold), mTOR-(P) (57%), p70S6K-(P) (11.7-fold), and ERK-(P) (66%) were observed in dystrophic mdx/utrn-/- muscle compared to wild type. Significant decreases in GSK-3-(P) (57%) and p38-(P)(2.9-fold) were also observed. Most of these signaling events were similar in dystrophic mice over-expressing the alpha7 integrin. However, a further increase in p70S6K-(P) (18-fold) and decrease in GSK-3-(P)(3.7-fold) were detected in alpha7BX2- mdx/utrn-/- compared to wild type mice and these changes were significant compared to mdx/utrn-/- mice. CONCLUSION: The alpha7beta1 integrin confers a protective effect in dystrophic mice and increased p70S6K activity appears important to this.

Candace M. Parchen1, Justin M. Percival2, Dao-Fu Dai3, Heidi N Gray1, Stanley C. Froehner2, Joseph A. Beavo1. 1Pharmacology, 2Physiology and Biophysics, 3Pathology, University of Washington, Seattle, WA

Duchenne muscular dystrophy (DMD) is the most prevalent type of muscular dystrophy and is the result of an X-linked mutation in the dystrophin gene. The progression of skeletal muscle damage is rapid in DMD patients and cardiomyopathy soon follows. We have investigated whether or not sildenafil citrate, a phosphodiesterase 5 (PDE5) inhibitor, can be used to ameliorate the age-related cardiac dysfunction in dystrophin-null (mdx) mice, a mouse model of DMD. Using echocardiography, we show that chronic sildenafil treatment improves several functional deficits in the cardiac performance of aged mdx mice. Collagen VI levels are also lower in the hearts of sildenafil-treated mdx mice, suggesting a remodeling of the extracellular matrix. This is the first study to report a cardioprotective effect of PDE5 inhibition in aged mdx mice. Overall, the data suggest that PDE5 inhibitors could be a useful treatment for the cardiomyopathy suffered by DMD patients.

Joseph M Vitale, Elizabeth Stillwell, Farah Khadim, Genie Elson, Aneela Altaf, Joel Schneider, Ghassan Yehia, Diego Fraidenraich. Cell Biology & Molecular Medicine, University of Medicine & Dentistry of New Jersey, Newark, NJ

Duchenne muscular dystrophy (DMD) is an incurable neuromuscular degenerative disease, caused by a mutation in the dystrophin gene. Mdx mice recapitulate DMD features. Here we show that injection of wild-type (WT) embryonic stem cells (ESCs) into mdx blastocysts produces mice with improved pathology. A small fraction of WT ESCs incorporates into the mdx mouse nonuniformly to upregulate protein levels of dystrophin in the skeletal muscle. The chimeric muscle shows reduced regeneration and restores dystrobrevin, a dystrophin-related protein, in areas with high and with low dystrophin content. WT ESC injection also normalizes the amount of fat, a tissue that does not express dystrophin. ESC injection without dystrophin does not prevent the appearance of phenotypes in the skeletal muscle or in the fat. Thus, dystrophin supplied by the ESCs reverses disease in mdx mice globally.

7) Murine mammary tumor growth is blunted in dystrophin deficient mdx mice

Mary Pat Meaney, Robert W. Grange, Young H. Ju. Human Nutrition, Foods and Exercise, Virginia Tech, Blacksburg, VA

Breast cancer (BC) is the second leading cause of cancer death in women in the US. Changes to the dystrophin glycoprotein complex (DGC), a multi-protein structure that likely plays mechanical and signaling roles, have been reported in BC cells. For example, expression of α- and β-dystroglycan appears to be inversely related to tumor stage. However, systemic absence of the DGC, as seen in the mdx mouse model of Duchenne muscular dystrophy, does not appear to induce spontaneous mammary tumor formation, indicating that presence of the DGC may be necessary for breast cancer initiation. We therefore hypothesized that breast tumor growth would be altered in mdx mice compared to wild type (C57BL/6) mice. We injected E0771 murine mammary adenocarcinoma cells into mdx and C57BL/6 mice. After 3 weeks of tumor growth, blood, skeletal muscles and tumors were collected and analyzed. Growth of E0771 tumors and serum content of migration and invasion chemokine markers, RANTES and MCP-1, was dramatically blunted in mdx mice. The dystrophin protein was not detectable in E0771 cells or tumors, suggesting that its expression, like that of other DGC components, may be suppressed or altered in this cancer cell line. The exact mechanism(s) of tumor inhibition in mdx mice is not presently known; however, our results suggest a mechanism for suppressing BC growth and progression may depend on the absence of one or more proteins in the DGC.