Ezrin Prompted Myoblast Differentiation and Muscle Fiber Specialization and Gastrocnemius Muscle Repair in Peroneal Nerve Injury Model through PKA-NFATs Signaling Pathway

Abstract Background: Muscular dystrophy is a destructive neuromuscular disease characterized by progressive muscle weakness and muscle atrophy. The role of Ezrin in myoblast differentiation/fusion and muscle atrophy is still unknown.Method: Gastrocnemius muscle atrophy model were prepared by mechanical clamp of peroneal nerve. Differentiating C2C12 cells treated with Ad-Ezrin or Ad-shEzrin were detected by gene chip, Q-PCR, immunofluorescence staining and Western blot.Results: Ezrin was expressed in MyHC I/II myofibers in vivo, and time-dependently increased during myoblast differentiation/fusion characterized by MyoG+/MEF2c nuclei and MyHC+ myotubes in vitro. Overexpression of Ezrin promoted myoblast differentiation/fusion in time-dependent manner, inducing the increased MyHC-I+ and MyHC-II+ muscle fiber specialization, the specific effects could be abolished by addition of Ad-Periaxin. Ad-Ezrin did not alter PKA and PKAreg II α levels, but PKAreg I α/β. The PKA inhibitor, H-89, remarkably abolished the over-expression effects by Ezrin on an increased myoblast differentiation/fusion. By contrast, Knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by the decreased PKA reg I/II ratio, the inhibitory effects could be eliminated by PKAreg I activator N6-Bz-cAMP. Meanwhile, Ad-Ezrin enhanced type I muscle fiber specialization, accompanied by the increased levels of NFATc1/c2.Furthermore, Ad-NFATc2 or Ad-NFATc4 reversed the inhibitory effects of Ad-shEzrin on myoblast differentiation/fusion. Importantly, in vivo transfection of Ad-Ezrin into gastrocnemius muscles in peroneal nerve injury model increased the numbers of MyHC-I+ and MyHC-II+ myofibers, reducing muscle atrophy and fibrosis.Conclusions: Ezrin activated PKA-NFAT-MyoD/MyoG/MEF2C signaling pathway, triggering myoblast differentiation/fusion and muscle fiber specialization in periaxin-depentdent manner, contributing to gastrocnemius muscles repair.