Myogenic program dysregulation is contributory to disease pathogenesis in spinal muscular atrophy

Abstract

Mutations in the survival motor neuron (SMN1) gene lead to the neuromuscular disease spinal muscular atrophy (SMA). Although SMA is primarily considered as a motor neuron disease, the importance of muscle defects in its pathogenesis has not been fully examined. We use both primary cell culture and two different SMA model mice to demonstrate that reduced levels of Smn lead to a profound disruption in the expression of myogenic genes. This disruption was associated with a decrease in myofiber size and an increase in immature myofibers, suggesting that Smn is crucial for myogenic gene regulation and early muscle development. Histone deacetylase inhibitor trichostatin A treatment of SMA model mice increased myofiber size, myofiber maturity and attenuated the disruption of the myogenic program in these mice. Taken together, our work highlights the important contribution of myogenic program dysregulation to the muscle weakness observed in SMA.

Figure 1.

Altered myogenic program and aberrant myotube formation in primary cells from Smn^2B/− mice. (A) Immunoblot analysis reveals decreased Smn, Pax7 and MyoD protein levels in proliferating primary myoblasts established from control and Smn^2B/− mice. Bar graph showing a significant decrease in Pax7 and MyoD protein levels. (B) Immunoblot analysis and quantification reveals significantly decreased levels of myogenic differentiation markers myogenin (MyoG) and myosin heavy chain (MHC) in cultured primary cells differentiated for 5 days. (C) Representative images of control and Smn^2B/− myoblasts differentiated into myotubes for 72 h and stained for MHC. Quantification of the number of nuclei per myotube revealed that there is a decrease in myoblast fusion in Smn^2B/− cells compared with controls. Scale bar = 50 μm. N = 3 for all experiments, *, P < 0.05; **, P < 0.01.

Figure 2.

Delayed expression of myogenic proteins in mouse models of SMA. (A) Representative immunoblots in hindlimb muscle from P0, P2 and P5 Smn^−/−;SMN2 mice revealed a robust decrease over time in Pax7, MyoD and myogenin protein levels compared with controls. The expression of MRF4 was unchanged at all time points. (B) Representative immunoblots showing aberrant expression of myogenic proteins at different time points in Smn^2B/− mice. The levels of myogenic factors are decreased in P2 muscles and increased at P21. At P6, MyoG protein levels were decreased. The levels of Pax7 and MyoD were high at P9 in Smn^2B/− compared with controls, while those of MyoG were low. Only Pax7 expression was mis-regulated at P15 compared with controls. (C) Time-course analysis of MyoD protein levels reveals a delay in the peak expression from P2 in control samples to P9 in Smn^2B/− muscle. N = 3 for all experiments.

Figure 3.

Decreased myofiber size and increased number of immature myofibers in muscle from mouse models of SMA. (A) Cross-sectional area measurements revealed smaller fibers in both mouse models at phenotype stage. Cross-sections of TA muscles from Smn^−/−;SMN2 and Smn^2B/− mice were taken at P5 and P21 respectively. (B) Representative images of hematoxylin and eosin stained cross-sections from control, prephenotype P2 Smn^−/−;SMN2, P2 and P9 Smn^2B/− mice, and phenotype stage P5 Smn^−/−;SMN2 and P21 Smn^2B/− TA muscles. Examples of myofibers with centrally located nuclei are depicted with arrows. Scale bars for P2 samples = 50 μm and scale bars for P5, P9 and P21 samples = 100 μm. (C) Quantification revealed an increased number of centrally located nuclei in both P2 and P5 Smn^−/−;SMN2 muscles. No change in the proportion of immature myofibers was observed at P2 in Smn^2B/− mice however, an increase in myofibers with centrally localized nuclei was observed in P9 and P21 samples. (D) Evan's blue dye (EBD) is taken up by degenerating fibers and is detected as a red fluorescence signal. Representative images demonstrating the absence of EBD uptake in muscle of control and phenotypic Smn^−/−;SMN2 and Smn^2B/− mice. TA muscle sections from the mdx muscular dystrophy mouse were used as a positive control for EBD uptake. Scale bar for P5 samples = 100 μm and the scale bar for P21 and mdx samples = 50 μm. N = 3 for all experiments. *, P < 0.05; **, P < 0.01.

Figure 4.

The myogenic program is altered even in muscles not subject to denervation in Smn^2B/− mice. (A) Representative images showing fully intact NMJs from cranial, TVA and RA muscles of control and P21 Smn^2B/− mice. Postsynaptic acetylcholine receptors were labeled with alpha-bungarotoxin (BTX, red) while the presynaptic terminal was labeled with antineurofilament (NF, green) and antisynaptic vesicle protein 2 (SV2, green). (B) Quantification of fully occupied endplates revealed differences between control and the TVA and RA muscles from Smn^2B/− mice but not for cranial muscles, suggesting that the latter are not subject to denervation. (C) Representative immunoblots showing altered protein levels of Pax7 and MyoD in innervated (cranial) and denervated (RA and TVA) muscles of Smn^2B/− mice. Interestingly, Pax7 and MyoD levels are unaffected in muscles following experimental denervation, at 1 or 7 days postsurgery. By comparison, a robust increase in myogenin levels can be detected as early as 1 day postdenervation and persists to 7 days following denervation. (D) Quantification analyses revealed differences in Pax7 and MyoD protein levels in all three muscle groups examined. The levels of myogenin however, were similar between control and Smn^2B/− mice for innervated and denervated muscles. (MyoG = myogenin, DEN = denervated). Scale bar = 20 μm. N = 3 for all experiments. *, P < 0.05; **, P < 0.01, ***, P < 0.001.

Figure 5.

TSA treatment improves myoblast fusion and muscle maturity in Smn^2B/− mice. (A) Representative images of Smn^2B/− myotubes treated with either vehicle (DMSO) or TSA. Myoblasts were treated for 24 h and then differentiated for 72 h and stained for myosin heavy chain (green) and 4′,6-diamidino-2-phenylindole (blue). (B) Quantification revealed that TSA treated Smn^2B/− cells had significantly more myotubes with five or more nuclei compared with Smn^2B/− controls. (C) Representative images of hematoxylin and eosin stained cross-sections of TA muscle from P25 Smn^2B/− mice treated with either DMSO or TSA. (D) Quantification revealed a significant decrease in the proportion of centrally located nuclei in Smn^2B/− muscles treated with TSA relative to those treated with DMSO. (E) Histogram demonstrating significantly fewer small caliber fibers and an increased proportion of large caliber fibers in muscles of Smn^2B/− animals following TSA treatment. (F) Quantification of RT-QPCR results demonstrating a significant decrease in transcript levels of embryonic (Emb) MHC and neonatal (Neo) MHC in P25 Smn^2B/− mice treated with TSA compared with Smn^2B/− mice treated with DMSO. (G) Smn^2B/− mice were treated for 3 days with either DMSO or TSA starting at P3. Immunoblot analyses demonstrating an increase in the protein levels of Pax7 and MyoD in TSA treated P6 Smn^2B/− compared with DMSO controls. (H) TSA treated Smn^2B/− mice showed increase in Pax7 positive cell numbers compared with DMSO controls. N = 3 for all experiments. Scale bars = 100 μm. *, P < 0.05; **, P < 0.01.