Insulin-like Growth Factor-1 Gene Transfer Augments Muscle IGF Protein Content Despite Cast Immobilization

Abstract

Insulin-like growth factor I (IGF-I) is a potent myogenic factor that has been shown to play a critical role in muscle regeneration and muscle hypertrophy. Viral-mediated gene transfer of IGF-I appears to facilitate muscle hypertrophy and improve muscle function, yet it is unclear whether the benefits of gene transfer are maintained during cast immobilization or with subsequent restoration of muscle function following cast immobilization. PURPOSE: To investigate the potential of virus-mediated gene transfer of IGF-1 to augment IGF-1 protein levels in skeletal muscle under normal loading/activity conditions and whether elevations are maintained during and after cast immobilization with subsequent reloading. METHODS: The anterior compartments of the left hindlimb of 43 young adult C57BL6 mice were injected with a recombinant adeno-associated virus vector for IGF-I (rAAV-1). The construct consisted of the entire rat IGF-I cDNA which encodes for IGF-I, a Myosin Light Chain (MLC) 1/3 promoter and enhancer, and SV40 polyadenlyation sequence. At 20 weeks of age, 35 of these mice were immobilized in a bilateral cast for 2 weeks with ∼60° of dorsiflexion to maximize anterior compartment muscle atrophy. After cast immobilization, the tibialis anterior muscle was removed from both legs at 0 days and 1,3,5, and 10 weeks of free cage reambulation. Eight mice remained uncasted for comparison. RT-PCr was performed to quantify IGF-1 mRNA levels. IGF-1 protein levels were quantified using a mouse IGF-1 immunoassay. RESULTS: IGF-1 mRNA levels were consistently elevated in injected limbs. In addition, there was a 27 fold increase in tibialis anterior IGF-1 protein content of the injected limb in uncasted mice (85.2+32.9ng/g injected; 3.2+0.48ng/g uninjected) (p<0.05). Significant differences in absolute values of IGF-1 protein content from injected to uninjected limbs persisted with cast immobilization and free cage reambulation (range: 14–19 fold differences) (p<0.05). When comparing the relative magnitude of side-to-side differences in IGF protein content across all timepoints, there was a significantly greater difference in IGF-1 protein content before cast immobilization (p<0.05). CONCLUSION: Muscle specific adeno-associated virus overexpression of IGF-I not only increased IGF-1 mRNA levels, but also resulted in markedly higher IGF-1 protein levels that remained elevated despite cast immobilization. Our findings suggest that gene transfer of IGF-1 may be able to guard skeletal muscle from the deleterious impact of immobilization/disuse and enhance the subsequent restoration of muscle function.