The Autophagy Regulatory Molecule CSRP3 Interacts with LC3 and Protects Against Muscular Dystrophy

Abstract

CSRP3/MLP (cysteine-rich protein 3/muscle Lim protein), a member of the cysteine-rich protein family, is a muscle-specific LIM-only factor specifically expressed in skeletal muscle. CSRP3 is critical in maintaining the structure and function of normal muscle. To investigate the mechanism of disease in CSRP3 myopathy, we performed siRNA-mediated CSRP3 knockdown in chicken primary myoblasts. CSRP3 silencing resulted in the down-regulation of the expression of myogenic genes and the up-regulation of atrophy-related gene expressions. We found that CSRP3 interacted with LC3 protein to promote the formation of autophagosomes during autophagy. CSRP3-silencing impaired myoblast autophagy, as evidenced by inhibited autophagy-related ATG5 and ATG7 mRNA expression levels, and inhibited LC3II and Beclin-1 protein accumulation. In addition, impaired autophagy in CSRP3-silenced cells resulted in increased sensitivity to apoptosis cell death. CSRP3-silenced cells also showed increased caspase-3 and caspase-9 cleavage. Moreover, apoptosis induced by CSRP3 silencing was alleviated after autophagy activation. Together, these results indicate that CSRP3 promotes the correct formation of autophagosomes through its interaction with LC3 protein, which has an important role in skeletal muscle remodeling and maintenance.

Keywords: CSRP3; apoptosis; atrophy; autophagy; myoblasts.

Figure 1

CSRP3 regulates mean muscle fiber and muscle mass in chicken skeletal muscles. (A and B) CSRP3 mRNA and protein expression in breast muscle after lentivirus injected. Hematoxylin and eosin (H&E) staining (C), mean muscle fiber CSA, scale bar = 100 μm (Black arrows point to necrotic myofibers) (D), and muscle weight (E) of breast muscle fiber cross-section after lentivirus injected. (F and G) Atrogin-1 and MuRF-1 mRNA expression and Atrogin-1 protein expression in breast muscle after lentivirus injected. Data are expressed as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 2

CSRP3 regulates the development of skeletal muscle myoblasts in chicken. (A and B) CSRP3 mRNA and protein in myoblasts transfected with si-CSRP3 of si-Ctrl. (C and D) The mRNA expression of MyoG, Mb, and MyHC, and protein abundance in myoblasts transfected with si-CSRP3 of si-Ctrl. (E and F) The mRAN and protein level of Atrogin-1 and MuRF-1 mRNA in myoblasts transfected with si-CSRP3 of si-Ctrl. Data are expressed as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 3

Gene expression analysis in ctrl and CSRP3 silenced cells by RNA sequencing. (A) Hierarchical clustering and heatmap of significant difference genes between chicken myoblasts transfected with control and CSRP3 siRNA. (B) Gene ontology (GO) enrichment analysis of significant differential expression genes between chicken myoblasts transfected with control and CSRP3 siRNA. (C) Pathway enrichment analysis of significant differential expression genes between chicken myoblasts transfected with control and CSRP3 siRNA.

Figure 3

Gene expression analysis in ctrl and CSRP3 silenced cells by RNA sequencing. (A) Hierarchical clustering and heatmap of significant difference genes between chicken myoblasts transfected with control and CSRP3 siRNA. (B) Gene ontology (GO) enrichment analysis of significant differential expression genes between chicken myoblasts transfected with control and CSRP3 siRNA. (C) Pathway enrichment analysis of significant differential expression genes between chicken myoblasts transfected with control and CSRP3 siRNA.

Figure 4

CSRP3 regulates autophagy in chicken myoblasts. (A) ATG5 and ATG7 mRNA expression in ctrl or CSRP3 siRNA cells. (B) Western blot analysis revealed LC3, P62, and Beclin-1 protein levels in ctrl and CSRP3 siRNA cells. (C) Immunofluorescence analysis of cell transfected with ctrl or CSRP3 siRNA then stained with LC3; scale bar = 25 μm. (D) The right diagram shows the average number of LC3 puncta in ctrl or CSRP3 siRNA cells. Data are expressed as mean ± SEM of three independent experiments. * p < 0.05, ** p < 0.01.

Figure 5

Co-localization of CSRP3 and LC3 in chicken myoblasts. (A) Representative confocal microscopy images of LC3 (red) and CSRP3 (green) were shown; scale bar = 20 μm. (B) Reciprocal co-immunoprecipitation analysis between CSRP3 and LC3 in ctrl and CSRP3 siRNA cells. Data are expressed as mean ± SEM of three independent experiments.

Figure 6

CSRP3 silence promotes cell apoptosis. (A) The ultrastructure of cells treated with 3MA, RAP or DMSO (ctrl) was observed by transmission electron microscopy. (B) Flow cytometry showed the apoptosis rates in control and CSRP3 siRNA cells. (C) The cleaved of caspase-3 and caspase-9 protein levels in control and CSRP3 siRNA cells. (D) Flow cytometry showed the apoptosis rates in control and CSRP3 siRNA cells incubated with 3MA. (E) Western blot analysis cleaved caspase-3 and caspase-9 protein levels in controls and CSRP3 siRNA cells incubated with 3MA. (F) Flow cytometry showed the apoptosis rates in control and CSRP3 siRNA cells untreated or treated with rapamycin (RAP). (G) The cleaved of caspase-3 and caspase-9 protein levels in CSRP3 siRNA cells untreated or treated with RAP. Data are expressed as mean ± SEM of three independent experiments.