The efficacy of follistatin to increase muscle mass and modulate the SMA phenotype was tested in hSMN2+/+
mice, referred to as SMA mice (33
). This model represents a relatively severe type of SMA with loss of lumbar motor neurons and progressive muscle weakness. SMA mice reach peak body weight at approximately post-natal day (p) 10 and die prematurely at around p16. Mice were genotyped using tail biopsy material at the day of birth (designated p1), and animals homozygous for Smn
loss were randomly assigned to treatment groups. To ensure that our delivery strategy resulted in an increase in muscle mass in the neonatal context of this relatively severe SMA mouse model, the wet weight of various muscles from follistatin-treated SMA mice was compared with that of vehicle-treated animals. Animals received intraperitoneal follistatin injections 0.75 mg/kg twice daily (b.i.d.). Gastrocnemius, tibialis anterior and triceps muscles from follistatin- and vehicle-treated animals were dissected at p6, p10 and p16. Muscles from wild-type mice were included for comparison. At p6, follistatin-treated triceps muscles were larger than vehicle-treated (Fig. A). This difference was even more pronounced at p10, when follistatin treatment resulted in significantly larger gastrocnemius and triceps muscles, approaching the weight of muscles from wild-type littermates (Fig. B). As expected, as disease progression advanced and the animals approached endstage, muscle weights were similar between phosphate-buffered saline (PBS)- and follistatin-treated animals (Fig. C). To account for potential variation in animal size, results are shown as muscle weights indexed to brain weights. Comparison of raw muscle weights gave virtually identical results (data not shown). To more closely analyze the muscle phenotype in treated SMA mice, muscle fiber size was examined at p6, p10 and p16 in the triceps brachii. Cross-sectional areas were not significantly different between vehicle- and follistatin-treated mice (Supplementary Material, Fig. S1
). This result was somewhat surprising as myostatin blockade has been shown to increase muscle fiber size (5
). However, transgenic over-expression of follistatin correlated to an approximate 1.6-fold increase in fiber area, with a 327% increase in muscle weight (5
). In our treatment model, we observe a more modest increase in muscle weight between 20 and 25%, and it is therefore likely that a proportional increase in fiber area would be too small to detect reliably. These data demonstrate that neonatal follistatin administration confers a temporal gain of muscle mass in SMA mice during the course of this rapidly developing neurodegenerative disease.
Figure 1. Muscle weights of SMA mice. Gastrocnemius (gastroc), tibialis antrior (TA) and triceps brachii (triceps) muscles were dissected at p6 (A), p10 (B), or p16 (C) and weighed. Pairs of muscle from the left and right sides of the animal were combined. Muscle (more ...)
To determine whether follistatin treatment affects the severity of the SMA phenotype, we measured time-to-right (TTR) for PBS- or follistatin-treated SMA pups. TTR was previously shown to be a sensitive measurement of gross motor function for these SMA animals (34
). To determine TTR, animals were placed on their backs, and the time required to turn upright was measured. Animals that failed to turn were recorded as 60 s. To examine a broader dosing range of recombinant follistatin, follistatin was administered at 10 mg/kg every other day (q.a.d.) and 1 mg/kg q.a.d., in addition to the initial dose of follistatin at 0.75 mg/kg b.i.d that increased muscle weight. These doses were administered q.a.d., assuming that the mice would better tolerate less frequent injections. TTR success rate was measured between p5 and p12 by adopting a turn/did-not-turn scoring system. Motor function of SMA mice was improved at all three doses tested, as a greater percentage of the follistatin-treated pups were able to right when compared with controls (Fig. A–C). In particular, the fraction of mice able to turn was statistically greater between p8 and p10 in the 0.75 mg/kg b.i.d. group. In addition, follistatin-treated animals tended to exhibit faster righting times (Fig. D–F). In Figure , p7 through p12 were emphasized due to the fact that about p12 all SMA animals, regardless of treatment, begin to right. Although there are clear statistical differences in the percentage of animals able to right following follistatin treatment, the severity of the disease likely impacts the ability to identify a statistically significant difference in the specific average righting times since only a small fraction of the vehicle-treated animals was able to turn at any given time point. The small sample size for the ‘vehicle-treated, but able to right’ group did not allow for a rigorous statistical analysis of the specific times to right.
Figure 2. Follistatin improves gross motor function. Mice were placed in a prone position and allowed to turn back to their feet within a maximum of 60 s. The capacity to turn within that time was plotted against age (A–C). Significant differences are indicated (more ...)
Follistatin treatment improved the capacity for overall ambulation in the SMA animals (Supplementary Material, Video S1
). The observed improvements in gross motor function were not accompanied by significant increases in body weight (Supplementary Material, Fig. S2
), which was not unexpected given the relatively modest size of neonatal muscles. Gross motor function increase with follistatin treatment (0.75 mg/kg b.i.d.) appears to be most pronounced from p6 to p12. These data directly correlate with the time frame of increased muscle mass (Fig. ). These findings demonstrate that administration of recombinant follistatin treatment is effective in lessening the severity of the SMA phenotype.
The SMA mice die prematurely following progressive muscle weakness, having an average lifespan of ~2 weeks (33
). To determine whether follistatin treatment extended survival of the SMA animals, lifespans for each of the three follistatin treatment groups were analyzed by Kaplan–Meyer survival curves. Although two groups did not exhibit significantly extended lifespans, the 1 mg/kg q.a.d. treatment increased mean lifespan by 4.6 days or ~30% (Fig. A–C). It is important to note that this change is seen not by altering the maximum lifespan, but rather by preventing the earlier deaths, thus an increase in the mean survival is observed. Additionally, the early deaths occurring in the vehicle-treated group were not observed in the 1 mg/kg q.a.d. treatment group. Although the basis for the different outcomes of these dosing regimens is currently unknown, these findings demonstrate that even in a relatively severe model of disease, follistatin administration can significantly extend survival of SMA mice. The expression of follistatin in muscles of a different model of muscle wasting, the SOD-1 ALS model, was recently shown to increase muscle mass but did not affect survival (23
), suggesting that the effects of follistatin on survival may be disease-specific.
Figure 3. Survival was determined using Kaplan–Meier curves. P-values were determined by log-rank (Walton–Cox) test (n = 10 for 10 mg/kg q.a.d., n = 15 for all other groups). To correct for Bonferroni interference, a stricter significance threshold (more ...)
As follistatin is a known inducer of muscle mass, we hypothesized that the lessening of disease severity was SMN-independent. Therefore, we examined SMN levels in tissues from follistatin-treated SMA animals. SMN levels were determined using p10 mice of the 0.75 mg/kg b.i.d. treatment group. This experimental group was selected on the basis of the phenotypic assessment, demonstrating that timed-righting and muscle weight differences were most pronounced at this stage. Tissues from p10 animals were dissected and analyzed by western blotting for SMN protein levels. As expected, in tissues from SMA mice, SMN levels were dramatically lower than those observed in wild-type tissues, as only low levels of full-length SMN are produced by the SMN2
transgene. Consistent with our hypothesis, follistatin treatment did not elevate SMN in spinal cord (Fig. A and B) or triceps muscle (Fig. C and D), and protein levels were statistically equivalent to those of the vehicle group. Plastin-3
was recently identified as a potential SMA-modifying gene, and its expression lessens the severity of the disease (32
). To determine whether the expression of this gene correlated with the lessening of the disease phenotype in follistatin-treated animals, plastin-3 levels were examined in the follistatin- and PBS-treated mice. Spinal cord protein extracts of p10 SMA pups were examined by western blot, demonstrating that plastin-3 levels were similar in follistatin-treated, PBS-treated pups and wild-type animals (Fig. E and F). These results demonstrate that the observed motor improvement was not correlated with changes in plastin-3 or full-length SMN levels.
Figure 4. Follistatin treatment does not affect SMA disease modifiers. SMN protein in p10 triceps (A and B) or upper spinal cord (C and D) lysates were examined by western blot. Both actin and IP90 are shown as loading controls. Graphs were quantified and average (more ...)
Several models of disease suggest a potentially protective interplay between both sides of the neuromuscular junction. In an ALS model, insulin-like growth factor I expression in muscle results in increased motor neuron survival (36
), whereas enhancing the functionality of the neuromuscular junction by maintaining the expression of post-synaptic proteins ameliorates the phenotype in a mouse model of muscular dystrophy (38
). Therefore, we speculated that enhanced muscle mass may lessen the severity of the SMA phenotype in part by preserving or maintaining neuronal circuitry. Spinal cord sections were analyzed to determine the number and size of ventral horn cell (VHC) numbers in follistatin-, PBS-treated and wild-type animals (Fig. A–C). The analysis of VHCs demonstrated that spinal cord sections from follistatin-treated animals contained approximately 180 VHCs when compared with 110 in vehicle controls (Fig. D). Although VHCs in follistatin-treated sections were not as abundant as in wild-type animals, they were significantly greater in numbers compared with vehicle-treated counterparts (P
< 0.02; Fig. D). Furthermore, the average cross-sectional area of the VHCs from follistatin-treated mice was larger than that of SMA mice treated with PBS and comparable with those of wild-type animals (Fig. E).
Figure 5. Analysis of VHCs of the lumbar spinal cord. Representative images of SMA vehicle (A), SMA follistatin (0.75 mg/kg b.i.d.) (B) and WT (C) spinal cords are shown of p10 animals. Lumbar VHCs were counted and the average number of VHCs per mouse (D) and the (more ...)
These results suggest that follistatin treatment can delay the death of spinal motor neurons in a relatively severe mouse model of SMA and that induction of known and potential modifiers of disease, SMN2 and plastin-3, are not obligate components of this process. We provide evidence that enhanced muscle mass in SMA improves gross motor function, which is consistent with the cellular analysis of treated animals, indicating a neural protective effect for VHCs. Taken together, our results demonstrate that follistatin treatment lessens the severity of the SMA phenotype and can increase mean survival in this relatively severe model of disease. The identification of an SMN-independent therapeutic target raises the possibility that combinatorial therapy aimed at increasing both muscle mass and SMN levels may prove beneficial.