Follistatin is commonly referred to as an activin-binding protein. Its primary goal is to inhibit myostatin, neutralize various TGF-beta compounds that are in charge of promoting growth and development, and improve homeostasis and regulation of the immune system. Follistatin-344 (FST-344) is an analogue of the naturally occurring peptide, follistatin, synthesized through the splicing of follistatin mRNA.The splicing of follistatin leads to two different isoforms FST-344 and FST-317 these then undergo further peptide cleavage to produce the variants FST-315 and FST-288, respectively. Many studies have been conducted determining the efficacy of myostatin inhibition of each follistatin isoform and how they relate to the degree of improvement of muscle growth [1].

Main Research Findings

1) Treatment with follistatin led to a dramatic increase in muscle weight of transgenic mice. This allowed researchers to conclude that follistatin is capable of effectively inhibiting myostatin, which ultimately resulted in significant muscle growth.

2) The results of this study determined that a single injection of FST-344 effectively inhibits myostatin in a way that promotes the enhancement of muscle size and strength. FST-344 was also successful at reversing degeneration and improving muscular function in mdx animals with induced DMD.

3) The research team of Rose et. Al concluded that treatment with follistatin enhances gross motor function and muscle size in rats exhibiting spinal muscular atrophy (SMA).

Selected Data

1) It has previously been determined that FST-344 acts as a myostatin antagonist by binding to TGF-beta; a compound that plays a crucial role in the regulation of skeletal muscle growth. Myostatin is found in the myotome compartment of somites before progressing to expression throughout the myogenic lineage. The inhibition of myostatin has been shown to increase skeletal muscle, researchers Lee examined the regulation of myostatin signaling in an attempt to identify myostatin inhibitors capable of promoting muscle growth.

Myostatin proteins were obtained from Chinese hamster ovary cell lines and from transgenic mice. It is important to note that many different species exhibit identical myostatin sequences in the biologically active C-terminal. This opens researchers up to further experimental opportunities regarding the efficacy of myostatin inhibition on muscle growth across species, primarily in human disease settings and livestock animals [2].

Because the C-terminal was found to be biologically active for several other TGF-beta compounds, the experiment continued by further purifying the C-terminal away from its propeptide. Additionally, many TGF-beta compounds are able to signal by binding to serine/threonine kinase receptors. The signaling pathway is triggered by the ligand binding to a type II receptor. In order to identify a correlation between myostatin and various type II receptors, the researchers crosslinked studies with radioiodinated myostatin C-terminal dimer on COS-7 cells that were transfected with expression constructs targeted for TGF-beta, BMP or activin type II receptors (Act RIIA or Act RIIB) [2].

2) The research team of Haidet et. Al sought to determine the potential of FST-344 and various other follistatin analogues to increase muscle mass in mice. AAVI viral particles were administered bilaterally into the quadriceps and tibialis anterior muscles of 4-week-old, wild-type C57BL/6 mice. After treatment with the myostatin inhibitors, the mice were observed and measured for muscle enlargement 725 days later.

Since the reported results of follistatin were so promising, researchers conducted a secondary test examining the efficacy of follistatin treatment in cases of muscle deterioration induced in the mdx mouse model of Duchenne muscular dystrophy (DMD). DMD is an X-linked recessive disease that results in loss of skeletal and cardiac muscle and function, and ultimately death [3].

The mdx animals were injected bilaterally in the quadriceps and tibialis muscles with either a low dose of 1 x 10^10 viral particles or a high dose of 1 x 10^11 viral particles. The injections took place as soon as the subjects reached 3 weeks of age and continued over the course of 5 months. The animals were then euthanized so the injected muscles could be dissected, weighed, snap-frozen, cryostat sectioned, and stained for analysis purposes. These same procedures were followed for the next experiment examining the effects of follistatin treatment when administered at an older age. In this case, the researchers chose to give the mdx animals the treatment 210 days after birth [3].

3) Researchers Rose et. Al attempted to determine the ability of follistatin to increase muscle mass, specifically in mice with the phenotype for spinal muscular atrophy (SMA) present. The mice in this model exhibited severe SMA with loss of lumbar function and progressive deterioration of muscles. The mice typically reached peak body weight 10 days after work and prematurely died 16 days after birth. On the day of birth the mice were genotyped via tail biopsy and separated into experimental treatment groups.

In order to ensure proper results the wet weight of various muscles of the mice treated with follistatin were compared to the wet weight of various muscles of the mice treated with a vehicle. The animals were intraperitoneally injected with a 0.75 mg/kg dose of follistatin, twice daily. Over the treatment period, portions of the gastrocnemius, triceps, and tibialis anterior muscles were dissected from the subjects on days 6, 10, and 16 after birth in order to observe changes in muscle size.

Additionally, the researchers examined whether follistatin is capable of affecting the SMA phenotype. Results were measured by a time-to-right (TTR) test in SMA pups treated with either active follistatin or a vehicle dose of PBS. The animals were flipped on their back while researchers measured the time it took for the subjects to flip over; if the animal was not able to flip over their time was recorded at 60 seconds. In order to determine an effective dose range of follistatin, the mice were administered either a 10 mg/kg or a 1 mg/kg dose of follistatin every other day. This was in addition to the original 0.75 mg/kg dose. After beginning treatment, TTR success rate was first tested on day 5 and again on day 12 in order to compare the performances of the mice in each experimental group [4].

Discussion

1) Results of the study were recorded through silver stain analysis of the myostatin protein that occurred after the initial round of purification. The analysis revealed the presence of two different protein species of 36- and 12.5-kDa. This data suggested that the purified myostatin protein contained a non-covalent complex of two propeptide compounds bound to the C-terminal. Western blot analysis found that the 36- and 12.5-kDa species were immunoreactive with antibodies that were raised against fragments of myostatin found throughout the propeptides and C-terminal.

After the initial experiment confirmed the bioactivity of the C-terminus, the researchers further purified the myostatin protein. The goal of this step was to purify myostatin away from its propeptides through the use of reverse-phase HPLC. The slices containing purified C-terminal were determined to be homogenous, while the slices enriched for the propeptide seemed to contain small fragments of the C-terminal. This is associated with high molecular weights and misfolded proteins [2].

Figure 1: Analysis of the purified myostatin protein following the heparin column or reverse-phase HPLC.

Furthermore, by crosslinking the studies the researchers were able to detect crosslinked complexes of receptors bound to myostatin that allowed for cells to express either Act RIIA or Act RIIB. Binding to Act RIIB was observed far more frequently than binding to Act RIIA. In order to determine the extent of activin type II receptor involvement in myostatin signaling, the researchers investigated the expression of a dominant-negative form of ACT RIIB in mice. This is a truncated form of Act RIIB that lacks the kinase domain that was placed downstream from a skeletal-muscle-specific myosin light chain promoter. Following pronuclear injections, researchers found seven animals positive for the transgene, all exhibiting significantly increased skeletal muscle mass, weighing up to 125% more than the control animals [2].

The researchers also decided to test the propeptide as well as follistatin in order to test their ability to block the effects of myostatin. Transgenic mice were generated in order to enhance the myosin light chain promoter used to drive expression of the myostatin antagonist. The independent transgenic mice exhibited a dramatic increase in muscle mass. In the first animal test subject, follistatin increased muscle weight by 194-327% in comparison to control animals. This jump in weight was due to a 66% increase in the number of fibers and a 28% increase in fiber diameter. The increase in muscle mass was comparable to mice with the myostatin knockout gene [2].

Figure 2: Muscle analysis; changes in fiber diameter and fiber quantity in transgenic mice.

2) After administering the viral particles of the myostatin blockers in the quadriceps and tibialis anterior muscles of mice, the researchers found that the animals given the myostatin inhibitors experienced a significant increase in body mass and an observable increase in muscle size. The greatest increase in muscle and body mass was seen in the animals treated with FST-344. These results were accompanied by an increase in hindlimb grip strength, and occurred without causing adverse effects on the cardiac muscle.

Figure 3: changes in body mass, muscle mass, and hindlimb grip strength

The secondary portion of the study regarding muscle deterioration in mdx mouse models with induced DMD also reported promising results. Administration of FST-344 led to increased muscle mass and decreased pathology. Both the high and low dose of FST-344 given to the subjects elicited an increase in circulating follistatin levels, however, the high dose resulted in a 15.3 +/- 2.1 ng/ml increase in follistatin serum levels while the low dose only increased levels by 6.8 +/- 0.4 ng/ml. Overall, the researchers concluded that muscle weight increased the most in animals administered FST-344. The results were not isolated to the injected muscles, results reported that remote sites experience myofiber hypertrophy.

Figure 4: staining of the tibialis anterior muscle reveals varying states of myofiber hypertrophy in muscles injected with follistatin or a control

The final portion of this experiment examined how FST-344 affects muscle strength in aged mdx animals. The mice were injected with follistatin 210 after birth and the research team began to observe increased muscle strength approximately 60 days post-injection. Muscle strength in these animals consistently increased over the 560 experimental period observed during this portion of the study. An interesting note of this study emphasized that the mdx animals started to experience degeneration as early as 180 days when left untreated. However, once they were injected with FST-344 on day 210, the animals experienced reversal of degeneration and improvement in strength. It was revealed that the increase in muscle mass seen in all mice injected with FST-344 related to an increase in hindlimb grip strength [3].

Figure 5: Changes in force produced in FST-344 treated aged animals

3) Follistatin was administered to mice experiencing SMA in order to see the effects of follistatin on muscle size and function. At 6 days after birth the tricep muscle treated with follistatin was larger than the tricep muscle in vehicle-treated animals. The size difference was even more pronounced at 10 days after birth; the growth was seen in both the gastrocnemius muscle and the triceps. On day 10 the weights of the muscle were approaching the same level as the wild-type control mice. The data collected allowed researchers to conclude that follistatin administration could be beneficial in the gain and maintenance of muscle mass in cases of SMA.

Figure 6: Muscle weight measured in the three different experimental groups on day 10 after birth.
The second portion of this study attempted to determine how follistatin treatment affects SMA of varying severity levels. This was measured through a TTR test conducted in pup treated with a vehicle or follistatin. All three doses given to the animals resulted in an enhancement in motor function in comparison to the control animals. There was a statistically significant increase in the percentage of mice able to turn over between day 8 after birth and day 10, specifically in the group receiving 0.75 ng/ml doses of follistatin.

Additionally, the animals treated with follistatin took a shorter amount of time to flip over than the other test subjects. By day 12 after birth the majority of the animals, regardless of SMA severity, were able to turn themselves over. While almost all animals were able to right themselves, there was significant variation in the time it took to complete the task dependent on severity levels. It should be noted that the benefits of follistatin treatment were not accompanied by increases in body weight or any severe adverse effects [4].

Figure 7: improvements in gross motor function in SMA mice treated with follistatin.

 

Conclusion

**LAB USE ONLY**
*This information is for educational purposes only and does not constitute medical advice. THE PRODUCTS DESCRIBED HEREIN ARE FOR RESEARCH USE ONLY. All clinical research must be conducted with oversight from the appropriate Institutional Review Board (IRB). All preclinical research must be conducted with oversight from the appropriate Institutional Animal Care and Use Committee (IACUC) following the guidelines of the Animal Welfare Act (AWA).

 

Citations

[1] Rodino-Klapac LR, Haidet AM, Kota J, Handy C, Kaspar BK, Mendell JR. Inhibition of myostatin with emphasis on follistatin as a therapy for muscle disease. Muscle Nerve. 2009 Mar;39(3):283-96. doi: 10.1002/mus.21244. PMID: 19208403; PMCID: PMC2717722.

[2] Lee SJ, McPherron AC. Regulation of myostatin activity and muscle growth. Proc Natl Acad Sci U S A. 2001 Jul 31;98(16):9306-11. doi: 10.1073/pnas.151270098. Epub 2001 Jul 17. PMID: 11459935; PMCID: PMC55416.

[3] Haidet AM, Rizo L, Handy C, Umapathi P, Eagle A, Shilling C, Boue D, Martin PT, Sahenk Z, Mendell JR, Kaspar BK. Long-term enhancement of skeletal muscle mass and strength by single gene administration of myostatin inhibitors. Proc Natl Acad Sci U S A. 2008 Mar 18;105(11):4318-22. doi: 10.1073/pnas.0709144105. Epub 2008 Mar 11. PMID: 18334646; PMCID: PMC2393740.

[4] Rose FF Jr, Mattis VB, Rindt H, Lorson CL. Delivery of recombinant follistatin lessens disease severity in a mouse model of spinal muscular atrophy. Hum Mol Genet. 2009 Mar 15;18(6):997-1005. doi: 10.1093/hmg/ddn426. Epub 2008 Dec 12. PMID: 19074460; PMCID: PMC2649020.

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