688. Adno-Associated Virus (AAV)-Mediated Catalase Overexpression Protects the Extensor Digitorum Longous Muscle from Contraction-Induced Injury

Abstract

Free radical injury is known to affect normal muscle contraction. Recent studies suggest that it also plays a critical role in the muscle aging process and in the pathogenesis of muscle diseases such as muscular dystrophy. Antioxidant gene therapy holds great promise in improving muscle function. Catalase decomposes hydrogen peroxide, a highly reactive oxygen species, to water and oxygen. In this study, we examined whether recombinant adeno-associated virus (rAAV)-mediated catalase over-expression could effect muscle contraction in the extensor digitorum longous (EDL) muscle in normal mice. To facilitate the transgene detection, we also engineered an HA tag in the C-terminal end of the catalase gene. Transfection of the AAV catalase proviral plasmid (pcisRSV.CatHA) in 293 cells yielded 2.4-fold higher catalase activity than that from a control proviral plasmid carrying the EGFP reporter gene. Western blot with both anti-catalase and anti-HA antibodies confirmed the ectopic catalase expression. To determine whether AV.RSV.CatHA can mediate efficient catalase expression in muscle, we delivered 1011 genome particles of AAV-7 catalase viruses to the left anterior tibialis (TA) muscle of 3-m-old BL10 mouse. The contra-lateral TA muscle was infected with an AAV-7 virus carrying the alkaline phosphatase reporter gene. Immunofluoresecnece staining and Western blot at one-month post-infection revealed high-level catalase expression in AV.RSV.CatHA infected muscle. The test whether catalase overexpression could enhance muscle function, we delivered 1011 genome particles of AAV-7 catalase viruses to the left EDL muscle of 3-m-old BL10 mice. The right EDL muscle was mocked infected with saline. At three months later, the catalase activity in AAV infected EDL muscle reached 1178 161 Ku/g while in saline injected muscle it only reached 463 42 Ku/g. The enhanced catalase activity in AAV infected muscle was further confirmed by zymogram assay. Despite AAV-mediated catalase overexpression, we did not detect any significant change in muscle weight, cross sectional area, the twitch and isometric tension. Forced lengthening during eccentric contraction has been shown to generate reactive oxygen species. To test whether AAV-mediated catalase overexpression could reduce free radical injury during eccentric contraction, we applied a total of 10 cycles of eccentric contraction on the EDL muscle. In mock-infected muscle, the isometric tension dropped to 92%, 69% and 56% of the starting level after the first, fifth and ninth eccentric contraction. While AV.RSV.CatHA infected muscle was significantly protected from the injury. The isometric tension only dropped to 96%, 74% and 62% of the starting levels, respectively. On average, catalase overexpression provided a 7.2% protection. In summary, our results suggest that AAV-mediated catalase over-expression represents a viable approach to reduce free radical injury and enhance muscle function.