Faculty of Basic Medical Sciences, Postgraduate Union Training Basement of Jin Zhou Medical University, Hubei University of Medicine, Shiyan, 442000, Hubei, People's Republic of China.
Department of Physiology, Hubei Key Laboratory of Embryonic Stem Cell Research,Faculty of Basic Medical Sciences, Hubei University of Medicine, Shiyan, 442000, Hubei, People's Republic of China.
J Transl Med. 2023 Mar 4;21(1):173. doi: 10.1186/s12967-023-04016-7.
Clinically, Charcot-Marie-Tooth disease (CMT)-associated muscle atrophy still lacks effective treatment. Deletion and mutation of L-periaxin can be involved in CMT type 4F (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 muscle atrophy by affecting the function of muscle satellite cells.
A gastrocnemius muscle atrophy model was prepared to mimic CMT4F and its associated muscle atrophy by mechanical clamping of the peroneal nerve. Differentiating C2C12 myoblast cells were treated with adenovirus-mediated overexpression or knockdown of Ezrin. Then, overexpression of L-periaxin and NFATc1/c2 or knockdown of L-periaxin and NFATc3/c4 mediated by adenovirus vectors were used to confirm their role in Ezrin-mediated myoblast differentiation, myotube formation and gastrocnemius muscle repair in a peroneal nerve injury model. RNA-seq, real-time PCR, immunofluorescence staining and Western blot were used in the above observation.
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 adenovirus vectors carrying Ezrin, but not Periaxin, into the gastrocnemius muscle in a peroneal nerve injury model increased the numbers of muscle myosin heavy chain (MyHC) I and II type myofibers, reducing muscle atrophy and fibrosis. Local muscle injection of overexpressed Ezrin combined with incubation of knockdown L-periaxin within the injured peroneal nerve or injection of knockdown L-periaxin into peroneal nerve-injured gastrocnemius muscle not only increased the number of muscle fibers but also recovered their 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 adenovirus vectors for knockdown of L-periaxin by shRNA. Overexpression of L-periaxin did not alter the inhibitory effects on myoblast differentiation and fusion mediated by knockdown of Ezrin by shRNA in vitro but decreased myotube length and size. Mechanistically, overexpressing Ezrin did not alter protein kinase A gamma catalytic subunit (PKA-γ cat), protein kinase A I alpha regulatory subunit (PKA reg Iα) or PKA reg Iβ levels but increased PKA-α cat and PKA reg II α levels, leading to a decreased ratio of PKA reg I/II. The PKA inhibitor H-89 remarkably abolished the effects of overexpressing-Ezrin on increased myoblast differentiation/fusion. In contrast, knockdown of Ezrin by shRNA significantly delayed myoblast differentiation/fusion accompanied by an increased PKA reg I/II ratio, and the inhibitory effects could be eliminated by the PKA reg activator N6-Bz-cAMP. Meanwhile, overexpressing Ezrin enhanced type I muscle fiber specialization, accompanied by an increase in NFATc2/c3 levels and a decrease in NFATc1 levels. Furthermore, overexpressing NFATc2 or knocking down NFATc3 reversed the inhibitory effects of Ezrin knockdown on myoblast differentiation/fusion.
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 muscle atrophy induced by nerve injury, especially in CMT4F.
临床上，Charcot-Marie-Tooth 病（CMT）相关肌肉萎缩仍然缺乏有效的治疗方法。L-周围蛋白缺失和突变可通过破坏髓鞘形成导致 CMT 型 4F（CMT4F），这可能与 Ezrin 在 L-周围蛋白自身聚合中的抑制作用有关。然而，L-周围蛋白和 Ezrin 是否通过影响肌卫星细胞的功能而独立或相互作用参与肌肉萎缩过程仍不清楚。
通过腓总神经机械夹闭制备腓肠肌萎缩模型，模拟 CMT4F 及其相关肌肉萎缩。用腺病毒介导的 Ezrin 过表达或敲低处理分化的 C2C12 成肌细胞。然后，用腺病毒载体过表达 L-周围蛋白和 NFATc1/c2 或敲低 L-周围蛋白和 NFATc3/c4，以确认它们在 Ezrin 介导的成肌细胞分化、肌管形成和腓总神经损伤模型中的腓肠肌修复中的作用。采用 RNA-seq、实时 PCR、免疫荧光染色和 Western blot 观察上述现象。
首次发现，L-周围蛋白瞬时表达在体外成肌细胞分化/融合过程中第 6 天最高，而 Ezrin 表达在第 4 天达到峰值。在腓总神经损伤模型中，腺病毒载体转导 Ezrin 而非 Periaxin 进入腓肠肌可增加肌球蛋白重链（MyHC）I 和 II 型肌纤维的数量，减少肌肉萎缩和纤维化。腓总神经损伤后局部肌肉注射过表达 Ezrin 结合敲低 L-周围蛋白，或向腓总神经损伤的腓肠肌注射敲低 L-周围蛋白，不仅增加了肌纤维数量，而且在体内恢复了其相对正常的大小。过表达 Ezrin 促进成肌细胞分化/融合，诱导增加的 MyHC-I 和 MyHC-II+肌纤维特化，并且可以通过添加腺病毒载体进行 shRNA 敲低 L-周围蛋白来增强特定作用。过表达 L-周围蛋白不会改变 Ezrin 通过 shRNA 敲低介导的成肌细胞分化和融合的抑制作用，但会降低肌管的长度和大小。在体外，过表达 Ezrin 不会改变 shRNA 敲低 Ezrin 对 PKA-γ cat、PKA Iα 调节亚基（PKA reg Iα）或 PKA reg Iβ 水平的抑制作用，但会增加 PKA-α cat 和 PKA reg IIα 水平，导致 PKA reg I/II 比值降低。PKA 抑制剂 H-89 显著消除了过表达 Ezrin 对增加成肌细胞分化/融合的作用。相反，shRNA 敲低 Ezrin 显著延迟成肌细胞分化/融合，同时伴有 PKA reg I/II 比值增加，PKA reg 激活剂 N6-Bz-cAMP 可消除其抑制作用。同时，过表达 Ezrin 增强了 I 型肌纤维特化，伴随着 NFATc2/c3 水平的增加和 NFATc1 水平的降低。此外，过表达 NFATc2 或敲低 NFATc3 逆转了 Ezrin 敲低对成肌细胞分化/融合的抑制作用。
Ezrin/Periaxin 表达的时空模式参与了成肌细胞分化/融合、肌管长度和大小以及肌纤维特化的控制，这与激活的 PKA-NFAT-MEF2C 信号通路有关，为神经损伤诱导的肌肉萎缩提供了一种新的 L-周围蛋白/Ezrin 联合治疗策略，特别是在 CMT4F 中。
