Ablation of specific insulin‐like growth factor I forms reveals the importance of cleavage for regenerative capacity and glycosylation for skeletal muscle storage

Abstract

Insulin‐like growth factor‐I (IGF‐I) facilitates mitotic and anabolic actions in all tissues. In skeletal muscle, IGF‐I can promote growth and resolution of damage by promoting satellite cell proliferation and differentiation, suppressing inflammation, and enhancing fiber formation. While the most well‐characterized form of IGF‐I is the mature protein, alternative splicing and post‐translational modification complexity lead to several additional forms of IGF‐I. Previous studies showed muscle efficiently stores glycosylated pro‐IGF‐I. However, non‐glycosylated forms display more efficient IGF‐I receptor activation in vitro, suggesting that the removal of the glycosylated C terminus is a necessary step to enable increased activity. We employed CRISPR‐Cas9 gene editing to ablate IGF‐I glycosylation sites (2ND) or its cleavage site (3RA) in mice to determine the necessity of glycosylation or cleavage for IGF‐I function in postnatal growth and during muscle regeneration. 3RA mice had the highest circulating and muscle IGF‐I content, whereas 2ND mice had the lowest levels compared to wild‐type mice. After weaning, 4‐week‐old 2ND mice exhibited higher body and skeletal muscle mass than other strains. However, by 16 weeks of age, muscle and body size differences disappeared. Even though 3RA mice had more IGF‐I stored in muscle in homeostatic conditions, regeneration was delayed after cardiotoxin‐induced injury, with prolonged necrosis most evident at 5 days post injury (dpi). In contrast, 2ND displayed improved regeneration with reduced necrosis, and greater fiber size and muscle mass at 11 and 21 dpi. Overall, these results demonstrate that while IGF‐I glycosylation may be important for storage, cleavage is needed to enable IGF‐I to be used for efficient activity in postnatal growth and following acute injury.