Single injection of AAV1-FS increases muscle mass and strength in young mdx mice. (a) Gross hindlimb muscle mass is increased in AAV1-FS-injected mdx animals at 180 days of age compared with AAV1-GFP-injected controls. (b) The mass of individual hindlimb and forelimb muscles is increased at 180 days of age in mice injected at 3 weeks of age with AAV1-FS compared with AAV1-GFP controls (n = 15). *, P ≤ 0.05. (c) Grip strength is improved in a dose-dependent manner in young mdx mice injected at 3 weeks of age with AAV1-FS followed for 180 days (n = 15). Red, high-dose AAV1-FS; blue, low- dose AAV1-FS; green, AAV1-GFP controls. Error bars represent standard errors.

Myostatin inhibitor proteins increase muscle mass and strength in wild-type C57BL/6 mice. (a) Gross hindlimb muscle mass is increased in all myostatin-inhibitor-protein treated mice at 725 days of age compared with AAV1-GFP injected controls. (b) Total body mass is significantly increased in AAV1-FS-injected (**, P ≤ 0.01) and AAV1-GASP-1-injected (*, P ≤ 0.05) mice compared with AAV1-GFP controls at 725 days of age (n = 10). (c) The mass of individual hindlimb and forelimb muscles is increased in mice injected with AAV expressing myostatin inhibitor proteins (n = 10). *, P ≤ 0.05. (d) Hindlimb grip strength improves >2 years in all treated mice with the greatest differences in AAV1-FS treated animals compared with AAV1-GFP controls (n = 10). Error bars represent standard error.

mdx mice treated with AAV1-FS show decreased markers of muscle damage and aged mdx mice are responsive to FS treatment with functional benefit. (a) Serum creatine kinase levels (units/liter) are decreased at 3 months after injection with AAV1-FS compared with AAV1-GFP-injected controls. (*, P < 0.05; n = 10.) Error bars represent standard errors. (b) Hindlimb grip strength is significantly increased (P ≤ 0.05) at 275 days and beyond in aged mdx mice treated with AAV1-FS at 210 days of age (n = 15). Red, high-dose AAV1-FS; green, AAV1-GFP controls. (c) H&E stain of aged gastrocnemius demonstrates reduced pathology when injected at 210 days of age with FS compared with GFP-injected controls. (Original magnification, ×40.) (d) H&E stained diaphragm of animals injected at 210 days of age with FS shows less fat replacement than GFP-injected controls at late stage. (Original magnification, ×20.)

mdx mice treated with AAV1-FS at 3 weeks of age and followed for 180 days demonstrate myofiber hypertrophy. (a) H&E staining of the tibialis anterior reveals myofiber hypertrophy in AAV1-FS injected muscle compared with AAV1-GFP control. (Original magnification, ×40.) (b) The mean diameter of dark (slow-twitch oxidative), intermediate (fast-twitch oxidative glycolytic), and light (fast twitch glycolytic) myofibers in the tibialis anterior (indicated by hatched line) is significantly increased in mice injected with AAV1-FS compared with AAV1-GFP-injected controls. (P < 0.001; n = 5). (c) The mean diameter of intermediate and light myofibers (indicated by hatched line) in the triceps is significantly increased in mice injected with AAV1-FS compared with AAV1-GFP-injected controls. (P < 0.001; n = 5.) (d) The distribution of dark, intermediate, and light fibers as determined by succinic dehydrogenase (SDH) staining is not changed by treatment with high or low doses of AAV1-FS. (P > 0.05 between all groups; n = 5.) (e) The mean number of fibers counted per an unbiased 0.14 mm² counting frame is decreased in the tibialis anterior of AAV1-FS-treated mice, given that the mean diameter of myofibers is increased. (*, P < 0.01; n = 5.) Error bars represent standard errors.