Reticulon-1C Involvement in Muscle Regeneration

Abstract

Skeletal muscle is a very dynamic and plastic tissue, being essential for posture, locomotion and respiratory movement. Muscle atrophy or genetic muscle disorders, such as muscular dystrophies, are characterized by myofiber degeneration and replacement with fibrotic tissue. Recent studies suggest that changes in muscle metabolism such as mitochondrial dysfunction and dysregulation of intracellular Ca²⁺ homeostasis are implicated in many adverse conditions affecting skeletal muscle. Accumulating evidence also suggests that ER stress may play an important part in the pathogenesis of inflammatory myopathies and genetic muscle disorders. Among the different known proteins regulating ER structure and function, we focused on RTN-1C, a member of the reticulon proteins family localized on the ER membrane. We previously demonstrated that RTN-1C expression modulates cytosolic calcium concentration and ER stress pathway. Moreover, we recently reported a role for the reticulon protein in autophagy regulation. In this study, we found that muscle differentiation process positively correlates with RTN-1C expression and UPR pathway up-regulation during myogenesis. To better characterize the role of the reticulon protein alongside myogenic and muscle regenerative processes, we performed in vivo experiments using either a model of muscle injury or a photogenic model for Duchenne muscular dystrophy. The obtained results revealed RTN-1C up-regulation in mice undergoing active regeneration and localization in the injured myofibers. The presented results strongly suggested that RTN-1C, as a protein involved in key aspects of muscle metabolism, may represent a new target to promote muscle regeneration and repair upon injury.

Keywords: Duchenne muscular dystrophy; RTN-1C; UPR; muscle differentiation.

Figure 1

(A) MyHC immunofluorescence images of C2C12 myotubes upon myogenic differentiation for 7 days. Scale bar: 20 μm. (B) Representative western blot and densitometric analysis of RTN-1C in C2C12 cells upon differentiation induction, by culture in DM for the indicated times. MyHC and myogenin were used as markers of differentiation process. Actin and tubulin were used as loading control. (n = 3; means ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001). (C) Representative western blot and densitometric analysis of RTN-1C and MyHC in C2C12 cells after transfection with RTN-1C vector. Ctr represents cells transfected with the empty vector. Tubulin was used as loading control. (n = 3; means ± SEM; * p < 0.05; ** p < 0.01). (D) Representative western blot and densitometric analysis of RTN-1C, MyHC and myogenin in C2C12 cells silenced for RTN-1C and cultured in DM for 72 h to induce differentiation. Scramble oligos were used as control. Tubulin was used as loading control. (n = 3; means ± SEM; * p < 0.05; ** p < 0.01).

Figure 2

(A) RTN-1C mRNA expression in anterior tibialis from WT and injured WT mice 3 and 7 days post injury (d.p.i.), measured by qPCR and normalized for GAPDH. (n = 3; means ± SEM; * p < 0.05). (B) Representative western blot and densitometric analysis of RTN-1C in tibialis muscles, from WT mice, subjected to injury and analyzed 3 d.p.i. Actin was used as loading control. (n = 8; means ± SEM; *** p < 0.001). (C,D) Representative western blot and densitometric analysis of RTN-1C in protein extracts from tibialis and diaphragm muscles of WT and mdx mice at 3 months of age. GAPDH was used as loading control. (n = 8; means ± SEM; ** p < 0.01; *** p < 0.001). (E) RTN-1C mRNA levels, quantified by qPCR and normalized for GAPDH, in tibialis muscles from WT and mdx mice at 3 months of age. (n = 8; means ± SEM; * p < 0.05).

Figure 3

(A) Representative western blot and densitometric analysis of RTN-1C in satellite cells, isolated from WT mice tibialis, exposed to differentiation medium for different time points. MyHC and myogenin were used as markers of the differentiation process. Tubulin was used as loading control. (n = 3; means ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001). (B) Representative western blot and densitometric analysis of RTN-1C, MyHC, and myogenin in satellite cells, isolated from WT mice, silenced for RTN-1C (siRTN-1C) and cultured in DM for 72 h to induce differentiation. Scramble oligos were used as control. Tubulin was used as loading control. (n = 3; means ± SEM; ** p < 0.01). (C) Representative western blot and densitometric analysis of RTN-1C expression in FACS-sorted satellite cells, analyzed after isolation in WT mice and injured mice at 3, 5, 7, and 15 days post injury (d.p.i.). (n = 3; means ± SEM; ** p < 0.01; *** p < 0.001). (D) Representative western blot and densitometric analysis of GRP78 and p-eIF2α in C2C12 cells upon differentiation induction, by culture in DM, for the indicated times. Actin was used as loading control. (n = 3; means ± SEM; * p < 0.05; ** p < 0.01; *** p < 0.001). (E) Representative western blot and densitometric analysis of GRP78 and p-eIF2α in protein extracts from tibialis and diaphragm muscles of WT and mdx mice at 3 months of age. GAPDH was used as loading control. (n = 8; means ± SEM; * p < 0.05; *** p < 0.001).