SMN Protein Contributes to Skeletal Muscle Cell Maturation Via Caspase-3 and Akt Activation

Abstract

Background/aim: In spinal muscular atrophy (SMA), systemic deficiency of survival motor neurons (SMN) caused by loss or mutation of SMN1 leads to SMA symptoms. SMA was, for a long time, considered as a selective motor-neuron disease. However, accumulated evidence suggests that skeletal muscle cells are affected by low levels of SMN protein. The purpose of this study was to elucidate the function of SMN protein in skeletal cell differentiation and maturation.

Materials and methods: In SMNΔ7 mice, which exhibit a systemic reduction of SMN protein, muscle atrophy was evaluated. To direct the effect of SMN against muscle cells, SMN functions were examined by knockdown of SMN in mouse myoblasts cell line C2C12 using siRNA.

Results: SMNΔ7 mice showed muscle atrophy accompanied by decreased both expression of a myogenesis marker and a proliferating marker. In SMN-knockdown myoblasts, early expression of myosin heavy chain and reduced multinuclear myotube formation were found. Decreased caspase-3 activity and reduced phosphorylation of Akt were observed at an early stage of differentiation in SMN-knockdown myoblasts.

Conclusion: A critical role of SMN protein in muscle cell differentiation via caspase-3 and Akt activation was shown.

Keywords: Myotube formation; skeletal muscles; spinal muscular atrophy; survival motor neurons.

Figure 1. Skeletal muscle pathology in spinal muscular atrophy (SMA) model mice. (A) Images of wild-type (WT) and SMNΔ7 mice at P11 which show intermediate symptoms, (B) Body weight of WT and SMNΔ7 mice at P11. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=3). **p<0.01 vs. WT mice (Student’s t-test). (C) Representative hematoxylin and eosin staining for gastrocnemius of WT and SMNΔ7 mice at P11. Scale bar=100 μm. (D) Mean fiber cross-sectional area. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=3). **p<0.01 vs. WT mice (Student’s t-test). (E) The distribution of fiber cross-sectional area. Data are presented as mean±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=3). (F) Representative fluorescence images of Ki67 in gastrocnemius of WT and SMNΔ7 mice at P11. Scale bar=75 μm. (G) Quantitative analysis of Ki67-positive cells. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=4). **p<0.01 vs. WT mice (Student’s t-test). (H) Representative fluorescence images of MyoD in gastrocnemius of WT and SMNΔ7 mice at P11. Scale bar=75 μm. (I) Quantitative analysis of MyoDpositive cells. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=4). *p<0.05 vs. WT mice (Student’s t-test).

Figure 1. Skeletal muscle pathology in spinal muscular atrophy (SMA) model mice. (A) Images of wild-type (WT) and SMNΔ7 mice at P11 which show intermediate symptoms, (B) Body weight of WT and SMNΔ7 mice at P11. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=3). **p<0.01 vs. WT mice (Student’s t-test). (C) Representative hematoxylin and eosin staining for gastrocnemius of WT and SMNΔ7 mice at P11. Scale bar=100 μm. (D) Mean fiber cross-sectional area. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=3). **p<0.01 vs. WT mice (Student’s t-test). (E) The distribution of fiber cross-sectional area. Data are presented as mean±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=3). (F) Representative fluorescence images of Ki67 in gastrocnemius of WT and SMNΔ7 mice at P11. Scale bar=75 μm. (G) Quantitative analysis of Ki67-positive cells. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=4). **p<0.01 vs. WT mice (Student’s t-test). (H) Representative fluorescence images of MyoD in gastrocnemius of WT and SMNΔ7 mice at P11. Scale bar=75 μm. (I) Quantitative analysis of MyoDpositive cells. Data are presented as means±S.E.M. (WT mice: n=4; SMNΔ7 mice: n=4). *p<0.05 vs. WT mice (Student’s t-test).

Figure 2. Altered expression of myosin heavy chain (MHC) and impaired fusion in SMN-knockdown C2C12 cells. (A) MHC and SMN protein expression was examined by western blot analysis in C2C12 cells. (B) Quantitative analysis of the expression level of SMN protein. Data are presented as means±S.E.M. (n=5 or 7), *p<0.05, **p<0.01 vs. Negative Control siRNA group (Welch’s t-test). (C) Quantitative analysis of the expression level of MHC. Data are presented as mean±S.E.M. (n=5 or 7), *p<0.05 vs. Negative Control siRNA group (Welch’s t-test). (D) Representative fluorescence image of MHC 6 days after transfection of siRNA. Scale bar=200 μm. (E) Quantitative analysis of the mean number of nuclei in each fiber. Data are presented as means±S.E.M. (n=8). **p<0.01 vs. Negative Control siRNA group (Student’s t-test).

Figure 3. Impaired caspase-3 activation during differentiation in SMNknockdown C2C12 cells. (A) Cleaved-caspase-3 and SMN protein expression was examined by western blot analysis in C2C12 cells. (B) Quantitative analysis of the expression level of cleaved-caspase-3. Data are presented as means±S.E.M. (n=5 or 7). **p<0.01 vs. Negative Control siRNA group (Student’s t-test).

Figure 4. Impaired Akt activation during differentiation in SMNknockdown C2C12 cells. (A) p-Akt and total Akt expression was examined by western blot analysis in C2C12 cells. (B) Quantitative analysis of the expression level of p-Akt and total Akt. Data are presented as means±S.E.M. (n=5 or 7). *p<0.05 vs. Negative Control siRNA group (Student’s t-test).