1. Center for Integrated Animal Genomics Research Experience in Molecular Biotechnology & Genomics Summer 2010 PGC1α over-expression rescues skeletal muscle from mdx mice Program supported by the National Science Foundation Research Experience for Undergraduates DBI-0552371 Connie M Santana¹, ², Delphine Gardan-Salmon ¹, Katrin Hollinger¹, Joshua T Selsby¹, Department of Animal Science, Iowa State University¹. Department of Biology, University of Puerto Rico, Rio Piedras Campus² Methods Results Conclusions [1] Angus, L.M. et al. (2005) Calceneurin-NFAT signaling, together with GABP amd peroxisome PGC-1α, drives utrophin gene expression at the neuromuscular junction. Am J Physiol. Cell Physiol. Vol. 289, p.p. C908-C917 [2] Davies,K.E and Nowak,K.J. (2006). Molecular mechanisms of muscular dystrophies: old and new players. Nature reviews. Molecular cell biology. pp. 762-773. [3] Liang H. and Ward,WF.(2006) PGC-1alpha: a key regulator of energy metabolism. Adv Physiol Educ.Vol. 30, p.p. 145-151. [4]Miura,P. and Jasmin, BJ.(2006). Utrophin upregulate for treating Duchenne or Becker muscular dystrophy:how close are we?.Elsevier p.p. 122-129. This work was partially supported by the National Science Foundation Research Experience for Undergraduates and the Center of Integrated Animal Genomics. I would like to thank Dr. Josh Selsby, Dr. Delphine Gardan, and Katrin Hollinger for their guidance, support, and help during this project. I would also like to thank Dr. Max Rothschild for the guidance given throughout the program. Introduction References Acknowledgements These data suggest that 3 wks of PGC-1α over-expression in mature dystrophin deficient mice:  reduced areas of hypercontracted cells, immune cell infiltration, and absent cells.  was successful in rescuing dystrophin deficient skeletal muscle. Together with previous data demonstrating disease prevention, these data demonstrating disease rescue strongly suggest that this pathway should be pursued as a potential therapeutic intervention.  Duchenne muscular dystrophy (DMD) is the most prevalent type of muscular dystrophy, affecting one in every 3,500 boys [4].  DMD is caused by an aberration in the dystrophin gene, which codes for the protein dystrophin [2].  Dystrophin is responsible for connecting the actin cytoskeleton ultimately to the extracellular matrix [2].  The absence of dystrophin permits the entrance of Ca 2+ to the sarcoplasm [2].  Increased Ca 2+ activates signaling cascades resulting in mitochondrial dysfunction, oxidative stress, and proteolysis leading to cell injury and death [2].  Utrophin, a dystrophin related protein, has been shown to function as a substitute for the missing dystrophin protein.  PGC-1α is a member of the transcription coactivators family that regulates mitochondrial function and the induction of slow gene expression, like utrophin.[1,3,4].  PGC-1α helps reduce the DMD pathology by increasing the expression of oxidative proteins, and promoting utrophin expression [2,3].  In dystrophin-deficient muscle, we have previously established that PGC-1a over-expression will prevent pathologic muscle changes as well as muscle injury caused by lengthening contractions, simulating normal activity [3]. Duchenne muscular dystrophy (DMD) is the most severe and common type of muscular dystrophy. Activation of peroxisome proliferator-activated receptor γ coactivator 1α (PGC-1α) is a possible therapeutic strategy for DMD treatment due to its involvement in mitochondrial biogenesis and regulation of utrophin expression, a dystrophy homolog. Previous studies have indicated the positive effects of PGC- 1α gene transfer in preventing typical pathology and acute injury in dystrophin-deficient muscles, however, the extent to which this treatment is effective in rescuing dystrophin- deficient skeletal muscle is unclear. In this experiment, mdx mice, a mouse model of DMD, were allowed to age for three weeks prior to being injected in the right hind limb with an adeno-associate virus 6 (AAV6) driving expression of PGC- 1α and in the left hind limb with a null virus. At six weeks of age, mice were euthanized and muscles removed. Over- expression of PGC-1α resulted in a reduction in total damaged area. More specifically, PGC-1a over-expression reduced areas of hypercontracted cells, immune cell infiltration, and absent cells, all indicators of muscle injury. Our results highlight the importance of PGC-1α in the rescue of dystrophin deficient skeletal muscles, and present a potential avenue for treating patients with DMD. Animal treatments: Three-week-old mdx mice were injected in the right hind limb with an AAV6 driving expression of PGC-1α (1x10 11 gc) and in the left hind limb with null AAV6. At six weeks of age the animals were sacrificed and muscles removed. Muscles were stored for histology or biochemistry, as appropriate. Histology: Upon being removed, muscles were placed in a freezing media and frozen in liquid nitrogen. In accordance with standard techniques, hematoxylin and eosin staining was performed. These sections were utilized for morphological assessments including determination of the area of immune cell infiltration, hypercontracted cells, and absent cells. Photoshop was used to reconstruct images of whole muscles and OpenLab software was used to analyse and quantify the area of the images. Statistical analysis: Treated and control mice were compared using paired T-tests, and the statistical significance was set at p<0.05. * indicates significant differences between these groups. Figure 1. Dystrophin-deficient mice were injected with virus causing over-expression of PGC-1α at three weeks of age and sacrificed at six weeks of age. PGC-1α over-expression caused a reduction in the muscle mass of the gastrocnemius but not in the soleus at time of sacrifice. * indicates p<0.05. Figure 2. Representative histological sections of dystrophin deficient soleus muscle (10x). Dystrophin-deficient mice were injected with an AAV driving PGC-1α (Right) or null virus (Left) at three weeks of age. Three weeks following injections we observed reduced total necrotic area in PGC-1 α expressing muscle when compared to null injected muscles (A). More specifically, areas of immune cell infiltration (B; Green arrows), absent cells (C; Black arrows), and hypercontracted cells (D; Blue arrows) were also reduced in PGC-1 overexpressing muscle compared to control limbs. * indicates significant different from control (p<0.05). Objective and Rationale The objective of this study focuses on determining the extent to which PGC-1α over-expression will rescue dystrophin- deficient skeletal muscle from disease associated injury. mdx mdx-PGC-1α A B C D Abstract