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Figure 8 Effect of rAAV/ASKN(KR) on Dystrophin Degradation and Calpain Activation

(A) Western blot analysis for -, ß-, and -sarcoglycan (SG) in hamster hearts 14 weeks after gene transfer. (B) Western blot analysis for dystrophin in hamster hearts 14 weeks after gene transfer. Full-length (arrow) and degraded (arrowheads) dystrophin were detected. (C) Western blot analysis for µ- and m-calpain. Arrows indicate autolyzed form of µ-calpain. (D) [Ca²⁺]_i after change to Na⁺-free solution. (Left panel) Changes in the ratio of fluorescent intensity of fura-2 at 340 nm to that at 380 nm after change to Na⁺-free solution. A representative averaged recording from at least 3 cardiomyocytes infected with AdV/LacZ or AdV/ASKN(KR) is shown. The right panel shows maximum fold changes in the fluorescent intensity ratio of fura-2 (n = 3). (E) Activation of ASK1 induced by Ca²⁺ overload. ASK1 activity was assessed by immune complex kinase assay with MKK6 as a substrate. (F) Calpain activity during Ca²⁺ overload. Left panel, changes in the fluorescent intensity of Boc-Leu-Met-CMAC (CMAC) during Ca²⁺ overload. A representative averaged recording from at least 3 cardiomyocytes is shown. To assess calpain-dependent protease activity, the cells were treated with or without calpeptin, a calpain inhibitor. Right panel shows slopes of CMAC fluorescent intensity curve between 10 and 30 min after change to Na⁺-free solution (n = 3). *p < 0.05 versus AdV/LacZ-infected cardiomyocytes. Abbreviations as in Figure 1.