1065. AAV2 Vector-Delivered Myostatin Propeptide Improves Muscle Healing after Injury

Skeletal muscle injuries are the most common injuries encountered in sports medicine. Muscle injuries can heal spontaneously through regeneration, but fibrosis impedes this process and results in incomplete functional recovery. We have demonstrated that TGF-β1 plays a significant role in both the initiation of fibrosis and the inducement of myofibrotic differentiation by myogenic cells in injured muscle. However, the recent identification of myostatin (MSTN), a member of the TGF-β superfamily, may yield new avenues for therapeutic intervention to improve muscle healing. MSTN is a potent negative regulator of muscle growth, and MSTN-deficient mice show dramatically increased skeletal muscle mass. We have found that the injured skeletal muscle of MSTN knockout (MSTN-/-) mice develops significantly less fibrous scar tissue and significantly larger regenerating myofibers than observed in normal wild-type (WT) control mice. We also found that MSTN stimulates proliferation and myofibrotic differentiation of fibroblasts in vitro. These results suggest that, like TGF-β1, MSTN may play a role in fibrosis. Myostatin propeptide (MPRO) inhibits MSTN's biologic activity by blocking the binding of MSTN to its receptor. In vivo overexpression of MPRO produces mice with increased muscle mass comparable to that observed in MSTN-/- mice. We hypothesized that use of MPRO in injured skeletal muscle would enhance muscle regeneration and inhibit fibrosis. In this study, we used an adeno-associated virus (AAV) vector to deliver the MPRO to myoblasts in vitro and direct injection of the virus to deliver the MPRO to the gastrocnemius muscle (GM) in vivo. In vitro, MPRO effectively neutralized MSTN and stimulated differentiation of C2C12 myoblasts. In contrast to uninfected C2C12 myoblasts, AAV2-MPRO-transduced myoblasts readily fused into larger myotubes containing numerous myonuclei. The addition of AAV2-MPRO led to a significant increase in the cells' fusion index in culture. We then injected the AAV2 vector carrying the MPRO gene into the GMs of adult BL6J mice; we injected the same amount of PBS into littermates as the control. Three weeks after AAV2 vector delivery, we lacerated both GMs of each mouse. Four weeks after vector injection, we observed larger-diameter regenerating myofibers in the GMs overexpressing MPRO than in the nontransduced GMs, although the difference was not significant. Masson's Trichrome histochemistry showed significantly less fibrous scar tissue in the AAV2-transduced GMs than in the control GMs. These findings are consistent with results from our previous studies showing that MSTN deficiency improves muscle healing after injury and show that the direct delivery of AAV2-MPRO into skeletal muscle is an effective and novel approach to improving muscle healing.