The spatiotemporal matching pattern of Ezrin/Periaxin involved in myoblast differentiation and fusion and Charcot-Marie-Tooth-associated muscle atrophy

Abstract Background Clinically, muscular dystrophy (MD), especially in Charcot-Marie-Tooth (CMT)-associated MD, still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT4F by destroying the myelin sheath form, which may be related to the inhibitory role of ezrin in the self-association of L-periaxin. However, it is still unknown whether L-periaxin and Ezrin are independently or interactively involved in the process of MD by affecting the function of muscle satellite cells. Method A gastrocnemius muscle (GA) atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells treated with Ad-ezrin or Ad-shezrin were detected by RNA-seq, Q-PCR, immunofluorescence staining and Western blotting. Ad-periaxin, Ad-shperiaxin, Ad-NFATc1/c2 or Ad-shNFATc3/c4 were used to confirm their role in ezrin-mediated myoblast differentiation, myotube formation and GA repair in a peroneal nerve injury model. Results For the first time, instantaneous L-periaxin expression was highest on the 6th day, while Ezrin expression peaked on the 4th day during myoblast differentiation/fusion in vitro. In vivo transduction of Ad-ezrin, but not Ad-ezrin, into the gastrocnemius muscle (GA) in a peroneal nerve injury model increased the numbers of MyHC-I+ and MyHC-II+ myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of Ad-ezrin combined with incubation of Ad-shperiaxin within the injured peroneal nerve or injection of Ad-shperiaxin into PNI-injured GA not only increased the number of muscle fibers but also recovered its size to a relatively normal level in vivo. Overexpression of Ezrin promoted myoblast differentiation/fusion, inducing increased MyHC-I+ and MyHC-II + muscle fiber specialization, and the specific effects could be enhanced by the addition of Ad-shPeriaxin. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by Ad-shEzrin in vitro but decreased myotube length and size. Mechanistically, Ad-Ezrin did not alter PKA-γ cat, PKA reg Iα and Iβ levels, but increase PKA-α cat and PKA reg II α levels, leading to the decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the overexpression effects of Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by a increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, Ad-ezrin enhanced type I muscle fiber specialization, accompanied by increase in NFATc2/c3 levels and decrease in NFATc1. Furthermore, Ad-NFATc2 or Ad-shNFATc3 reversed the inhibitory effects of Ad-shEzrin on myoblast differentiation/fusion. Conclusions The spatiotemporal pattern of Ezrin/Periaxin expression was involved in the control of myoblast differentiation/fusion, myotube length and size, and myofiber specialization, which was related to the activated PKA-NFAT-MEF2C signaling pathway, providing a novel L-Periaxin/ezrin joint strategy for the treatment of MD induced by nerve injury, especially in CMT4F.