Figure 4

Muscle recovery from cardiotoxin injury requires SSPN-dependent Akt activation. (A) An acute injury model was used to investigate muscle repair in wild-type and SSPN-null mice. Quadriceps injected with equivalent volumes of saline (mock) and cardiotoxin dissolved in saline (CTX) were evaluated. CTX causes localized regions of myofiber necrosis followed by regeneration (for review, [125]). Mice at six weeks of age were injected with CTX (or mock) and analyzed after seven days. To test the dependency of muscle repair on Akt signaling, a subset of mice was pre-treated with adenovirus containing constitutively active Akt (Ad-caAkt) two days prior to CTX treatment. (B) Analysis was performed using H&E, laminin/eMHC, and laminin/EBD stained sections from quadriceps muscle. SSPN-deficient mice exhibit increased active regeneration (eMHC, 30%) seven days after CTX injury when wild-type mice have already undergone successful repair (black). Administration of Ad-caAkt prior to CTX treatment rescued the repair defect in SSPN-deficient mice (grey). (C) Immunoblot analysis of RIPA quadriceps lysates revealed reductions in the levels of dystrophin (Dys), utrophin (Utr), integrin (β1 integrin), phosphorylated Akt (P-Akt), and phosphorylated p70S6K (P-p70S6K) in SSPN-null mice compared to wild-type. Quantification of utrophin and P-Akt is provided. Coomassie blue (CB) serves as a loading control. (D) Immunoblot analysis of RIPA lysates from quadriceps pre-treated with Ad-caAkt revealed reductions in the levels of dystrophin (Dys) and integrin (β1 integrin) in SSPN-null mice compared to wild-type. Utrophin levels were restored in SSPN-null mice compared to wild-type mice with supplementation of active Akt (right). These data reveal that SSPN is upstream of the Akt signaling pathway regulating utrophin expression. The Ad-caAkt contains the HA-tag for detection [19]. RIPA, radioimmunoprecipitation assay.