Inhibition of myostatin does not ameliorate disease features of severe spinal muscular atrophy mice

Abstract

There is currently no treatment for the inherited motor neuron disease, spinal muscular atrophy (SMA). Severe SMA causes lower motor neuron loss, impaired myofiber development, profound muscle weakness and early mortality. Myostatin is a transforming growth factor-beta family member that inhibits muscle growth. Loss or blockade of myostatin signaling increases muscle mass and improves muscle strength in mouse models of primary muscle disease and in the motor neuron disease, amyotrophic lateral sclerosis. In this study, we evaluated the effects of blocking myostatin signaling in severe SMA mice (hSMN2/delta7SMN/mSmn(-/-)) by two independent strategies: (i) transgenic overexpression of the myostatin inhibitor follistatin and (ii) post-natal administration of a soluble activin receptor IIB (ActRIIB-Fc). SMA mice overexpressing follistatin showed little increase in muscle mass and no improvement in motor function or survival. SMA mice treated with ActRIIB-Fc showed minimal improvement in motor function, and no extension of survival compared with vehicle-treated mice. Together these results suggest that inhibition of myostatin may not be a promising therapeutic strategy in severe forms of SMA.

Figure 1.

SMA disease progression is associated with increased follistatin gene expression and decreased myostatin gene expression. Follistatin and myostatin transcripts were measured in all hind-limb muscles at different ages in SMA and CL mice and values were normalized for each transcript to the P13 CL value. Adult mice were approximately 6 weeks old [*P < 0.05, **P < 0.001, n = 5 mice per group except for P1 SMA (n = 3), P5 SMA (n = 3) and P9 CLs (n = 4)].

Figure 2.

Overexpression of follistatin does not substantially increase muscle mass in SMAΔ7 mice. (A) The extent of follistatin transgene expression was determined by qRT–PCR in P10 quadriceps and gastrocnemius muscles from follistatin-negative and -positive mice [*P < 0.005, n = 3 per group (2 CL and 1 SMA)]. (B) Weights of gastrocnemius (gastroc), quadriceps (quad) and triceps muscles in P10 follistatin-negative and -positive SMA mice. Only the quadriceps showed a significant trend of increased muscle weight in follistatin-positive SMA mice (P = 0.065). (C) Hematoxylin and eosin (H&E) stained cross sections of quadriceps muscle from a P10 SMA mouse (left) and a P10 follistatin-positive SMA mouse.

Figure 3.

Overexpression of follistatin does not improve disease features of severe SMA mice. (A) Average total righting time in follistatin-negative (n = 8) and follistatin-positive SMA mice (n = 17). (B) Kaplan–Meier survival curves comparing follistatin-negative and follistatin-positive SMA mice. Median survival in follistatin-negative SMA mice was 13 and median survival in follistatin-positive mice was 14 (Log rank P = 0.6, n = 11 follistatin-negative, 25 follistatin-positive SMA mice).

Figure 4.

ActRIIB-Fc increases muscle mass and decreases fat mass in neonatal mice. CLs were treated with ActRIIB-Fc for 2 weeks. Muscle (left) and fat (right) weights from CL mice treated with vehicle (n = 6) or ActRIIB-Fc (n = 5) (*P < 0.05, **P < 0.005).

Figure 5.

ActRIIB-Fc does not extend survival of SMA mice. (A) Average total righting time, (B) average position score, (C) total number of pulls, (D) total latency to fall time on the hind-limb suspension test, (E) weights and (F) Kaplan–Meier survival curves of vehicle-treated (n = 21) and ActRIIB-Fc-treated SMA mice (n = 20) (Log rank P = 0.008).