RNA transcript expression of IGF-I/PI3K pathway components in regenerating skeletal muscle is sensitive to initial injury intensity

Objective: Skeletal muscle regeneration is a complex process involving the coordinated input from multiple stimuli. Of these processes, actions of the insulin -like growth factor-I (IGF-I) and phosphoinositide 3-Icinase (PI3K) pathways are vital; however, whether IGF-I or PI3K expression is modified during regeneration relative to initial damage intensity is unknown. The objective of this study was to determine whether mRNA expression of IGF-I/P13K pathway components was differentially regulated during muscle regeneration in mice in response to traumatic injury induced by freezing of two different durations. Design: Traumatic injury was imposed by applying a 6-mm diameter cylindrical steel probe, cooled to the temperature of dry ice (-79 degrees C), to the belly of the left tibialis anterior muscle of 12-week-old C57BL/6J mice for either 5 s (5 s) or 10 s (10 s). The right leg served as the uninjured control. RNA was obtained from injured and control muscles following 3, 7, and 21 days recovery and examined by real-time PCR. Expression of transcripts within the IGF, PIN, and Akt families, as well as for myogenic regulatory factors and micro-RNA5 were studied. Results: Three days following injury, there was significantly increased expression of Igf1, Igf2, Igf1r,lgf2r,Pik3cb, Pik3cd, Pik3cg, Pik3r1, Pik3r5, Aktl, and Akt3 in response to either 5 s or 10 s injury compared to uninjured control muscle. There was a significantly greater expression of Pik3cb, Pik3cd, Pik3cg, Pik3r5, Akt1, and Akt3 in 10 s injured muscle compared to 5 s injured muscle. Seven days following injury, we observed significantly increased expression of Igfl, Igf2, Pik3cd, and Pik3cg in injured muscle compared to control muscle in response to 10 s freeze injury. We also observed significantly reduced expression of Igflr and miR-133a in response to 5 s freeze injury compared to control muscle, and significantly reduced expression of Ckm, miR-1 and miR-133a in response to 10 s freeze injury as compared to control. Twenty-one days following injury, 5 s freeze -injured muscle exhibited significantly increased expression of Igf2, Igf2r, Pik3cg, Akt3, Myod1, Myog, Myf5, and miR-206 compared to control muscle, while 10 s freeze -injured muscles showed significantly increased expression of Igf2, Igf2r, Pik3cb, Pik3cd, Pik3r5, Akt1, Akt3, and Myog compared to control. Expression of miR-1 was significantly reduced in 10 5 freeze -injured muscle compared to control muscle at this time. There were no significant differences in RNA expression between 5 s and 10 s injury at either 7 d or 21 d recovery in any transcript examined. Conclusions: During early skeletal muscle regeneration in mice, transcript expressions for some components of the IGF-I/PI3K pathway are sensitive to initial injury intensity induced by freeze damage. Published by Elsevier Ltd.