Increased IGF-1 in muscle modulates the phenotype of severe SMA mice

Abstract

Spinal muscular atrophy (SMA) is an inherited motor neuron disease caused by the mutation of the survival motor neuron 1 (SMN1) gene and deficiency of the SMN protein. Severe SMA mice have abnormal motor function and small, immature myofibers early in development suggesting that SMN protein deficiency results in retarded muscle growth. Insulin-like growth factor 1 (IGF-1) stimulates myoblast proliferation, induces myogenic differentiation and generates myocyte hypertrophy in vitro and in vivo. We hypothesized that increased expression of IGF-1 specifically in skeletal muscle would attenuate disease features of SMAΔ7 mice. SMAΔ7 mice overexpressing a local isoform of IGF-1 (mIGF-1) in muscle showed enlarged myofibers and a 40% increase in median survival compared with mIGF-1-negative SMA littermates (median survival = 14 versus 10 days, respectively, log-rank P = 0.025). Surprisingly, this was not associated with a significant improvement in motor behavior. Treatment of both mIGF-1(NEG) and mIGF-1(POS) SMA mice with the histone deacetylase inhibitor, trichostatin A (TSA), resulted in a further extension of survival and improved motor behavior, but the combination of mIGF-1 and TSA treatment was not synergistic. These results show that increased mIGF-1 expression restricted to muscle can modulate the phenotype of SMA mice indicating that therapeutics targeted to muscle alone should not be discounted as potential disease-modifying therapies in SMA. IGF-1 may warrant further investigation in mild SMA animal models and perhaps SMA patients.

Figure 1.

Overexpression of mIGF-1 results in increased muscle mass in SMA mice. (A) IGF-1 and (B) IGFR1 mRNA expression levels were determined by qRT–PCR in quadriceps muscles from P10 mIGF-1^NEG and mIGF-1^POS SMA and heterozygous (Het) littermate mice [*P < 0.05, **P < 0.01 and ***P < 0.001, n = 6 mice per group except for SMA mIGF-1^NEG (n = 7)]. (C) H&E stained cross-sections of quadriceps muscle from P10 SMA mIGF-1^NEG (left) and a P10 mIGF-1^POS SMA mice (right). Scale bar = 50 µm. (D) Weights of gastrocnemius (gastroc), quadriceps (quad) and triceps muscles in P10 mIGF-1^NEG and mIGF-1^POS SMA mice (*P < 0.05, n = 7 mIGF-1^NEG and n = 8 mIGF-1 + SMA mice). (E) Myofiber diameter in the P10 quadriceps muscle in SMA mIGF-1^NEG and SMA mIGF-1^POS mice (*P < 0.05, n = 3 mIGF-1^NEG, and n = 4 mIGF-1^POS SMA mice). Values represent average ± SEM.

Figure 2.

Expression of SMN is not changed by mIGF-1 overexpression. (A) SMN678 and SMN68 mRNA levels were determined in quadriceps isolated from P10 SMA mIGF-1^NEG (n = 7) and SMA mIGF-1^POS mice (n = 6). (B) Embryonic, perinatal and adult MyHC and (C) AChR γ and AChR ε mRNA levels were measured in the same muscles (*P < 0.05). Values represent average ± SEM.

Figure 3.

Overexpression of mIGF-1 modestly improves survival of SMA mice. (A) Kaplan–Meier survival curves comparing mIGF-1^NEG and mIGF-1^POS SMA mice. Median survival in mIGF-1^NEG SMA mice was 10 days and median survival in mIGF-1^POS mice was 14 days (log-rank P = 0.025, n = 52 mIGF-1^NEG and n = 33 mIGF-1^POS SMA mice). (B) Body weight was increased in SMA mIGF-1^POS mice on P7, P8 and P9 compared with SMA mIGF-1^NEG mice (*P < 0.05 and **P < 0.01, as determined by multiple imputation test). (C) Average righting time showed no difference between SMA mIGF-1^NEG and mIGF-1^POS mice (n = 52 and n = 33, respectively). (D) The composite tube test score also did not show statistically significant differences between mIGF-1^NEG and mIGF-1^POS SMA mice (n = 8 mice per group). Values represent average ± SEM.

Figure 4.

The benefits of TSA and mIGF-1 are not synergistic. (A) Kaplan–Meier survival curves comparing mIGF-1^NEG and mIGF-1^POS SMA mice receiving TSA. Median survival in vehicle-treated mIGF-1^NEG mice was 14 days (n = 9) and in vehicle-treated mIGF-1^POS mice was 17 days (n = 9) (P = 0.94). TSA treatment increased median survival of mIGF-1^NEG mice (n = 7) to 22 days (P = 0.059) and of mIGF-1^POS mice (n = 8) to 25 days (P < 0.005). (B) Body weights were increased in SMA mice receiving TSA compared with SMA mice receiving vehicle, but mIGF-1 did not result in a further increase as determined by the multiple imputation analysis test (vehicle mIGF-1^NEG versus TSA mIGF-1^NEG: *P < 0.05 and **P < 0.01 and vehicle mIGF-1^POS versus TSA mIGF-1^POS: ^#P < 0.05 and ^##P < 0.01). (C) Righting time showed a trend toward improvement in TSA treated mice, but was not further increased by mIGF-1. Values represent average ± SEM.