Drug Discovery and Translational MedicineFollistatin: A Novel Therapeutic for the Improvement of Muscle Regenerations
ABSTRACT
Follistatin (FST) is a member of the tissue growth factor β family and is a secreted glycoprotein that antagonizes many members of the family, including activin A, growth differentiation factor 11, and myostatin. The objective of this study was to explore the use of an engineered follistatin therapeutic created by fusing FST315 lacking heparin binding activity to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) as a systemic therapeutic agent in models of muscle injury. Systemic administration of this molecule was found to increase body weight and lean muscle mass after weekly administration in normal mice. Subsequently, we tested this agent in several models of muscle injury, which were chosen based on their severity of damage and their ability to reflect clinical settings. FST315-ΔHBS-Fc treatment proved to be a potent inducer of muscle remodeling and regeneration. FST315-ΔHBS-Fc induced improvements in muscle repair after injury/atrophy by modulating the early inflammatory phase allowing for increased macrophage density, and Pax7-positive cells leading to an accelerated restoration of myofibers and muscle function. Collectively, these data demonstrate the benefits of a therapeutically viable form of FST that can be leveraged as an alternate means of ameliorating muscle regeneration.
Introduction
A multitude of diseases affect skeletal muscle, ranging from direct injury to the soft tissue or nerve supply, chronic inflammation, aging, and cancer. Irrespective of the mode, it results in the same outcome: limited mobility with the contemporaneous loss of muscle function and mass. Skeletal muscle injuries are one of the most common types of work- and sports-related injuries that present a major challenge to primary care physicians, physical therapists, and sports medicine professionals. Individuals can sustain muscle injuries through a myriad of different mechanisms, including improper handling/biomechanics of heavy materials (e.g., muscle strain, tears, neuronal impingement, and pulls), trauma (falls, surgeries, and sports collisions), and genetic disorders (muscular dystrophy). Patients who suffer from these types of occurrences have very few options with regards to treatment, which limits their ability to return to work or resume their current lifestyle. To mitigate the long-term issues with muscle injury/trauma, the current conservative treatment incorporates the minimization of inflammation with limb elevation and local cooling, in conjunction with a regimented physical therapy plan and an adjuvant treatment with nonsteroidal anti-inflammatory drugs. Irrespective of complying with the aforementioned treatment protocol, it can be difficult to achieve complete recovery from injury because of concomitant development of fibrosis that is most often seen due to comorbidities associated with metabolic and structural aging.
Myostatin or growth differentiation factor 8 (Gdf8) is a well-studied member of the tissue growth factor β (TGFβ) family that regulates muscle mass through development and postnatally. Gdf8 has been the center of focus in developing therapeutics, which inhibit and neutralize its activity. A variety of methods to accomplish Gdf8 inhibition include the following: specific antibodies which govern the ability of the ligand to interact with its receptor, dominant negative-mimetic proteins, adenoviruses to deliver a propeptide inhibitor, and a receptor decoy composed of the ectodomain of the activin receptor IIB conjugated to the Fc domain of the IgG molecule (Teo et al., 2012).
Another TGFβ superfamily member, follistatin (FST), has also been found to play a significant role in the management of skeletal muscle size and mass (Amthor et al., 2004; Lee, 2007). FST is composed of a 63-residue amino (N)-terminal domain, followed by three successive follistatin domains of 73–75 amino acids termed FSD1, FSD2, and FSD3 (Wang et al., 2000; Cash et al., 2009). There are three reported isoforms that exist: FST288, FST315, and FST300 (or FST303), which are thought to be products of post-translational modification events (Hashimoto et al., 2000; Sidis et al., 2006). FST288 and 315 proteins differ only in their possession of exon 6, which has been shown to influence their interactions with heparin, whereas FST303 is produced by proteolytic processing of the C-terminal tail domain (Sugino et al., 1993). Full-length exon 6 reduces the overall binding to cell surface heparin-sulfated proteoglycans, which is observed with the FST315 isoform, whereas FST288 lacks exon 6 and, therefore, displays greater cell surface affinity (Sidis et al., 2006). Affinity to cell surface receptors helps dictate the general biological actions of FST with FST288 conducting itself more in an autocrine fashion and FST315 (primarily found in circulation) behaving more as an endocrine factor (Bilezikjian et al., 2004; Yuen and Ge, 2004; Kumar, 2005).
FST proteins were found to associate with Gdf8 in the serum and were later demonstrated to antagonize Gdf8-mediated inhibition of myogenesis (Thomas et al., 2000; Hill et al., 2002). FST-transgenic mice produced a robust skeletal muscle hypertrophy phenotype and, when additionally crossed in a Gdf8-null mouse, resulted in a quadrupling effect on muscle (Lee, 2007). This finding indicated the existence of other muscle regulators besides Gdf8 that follistatin can modulate. Activin A has been the logical option for the second player in regulating muscle mass, and recent data from several laboratories would confer this as the best choice (Gilson et al., 2009; Lee et al., 2012).
Finally, the main objective of this study was to test a newly engineered FST biotherapeutic that fuses a heparin-deficient binding form of follistatin 315 to the N terminus of a murine IgG1 Fc (FST315-ΔHBS-Fc) (Sidis et al., 2005; Datta-Mannan et al., 2013) in multiple models of muscle injury. Our results clearly demonstrate the viability of a systemically administered form of FST that can increase muscle mass, facilitate muscle repair, and restore muscle function in multiple models of muscle injury. Combining FST’s effects on inflammation and pro-regenerative capabilities makes it a practical option for the treatment of muscle diseases where injury is associated.
Access through your organization
Check access to the full text by signing in through your organization.Access through your organization
Section snippets
Animal Care and Use
All mouse experiments were performed with the approval of Eli Lilly and Company’s Institutional Animal Care and Use Committee and are in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.
For all studies described here, 10-week-old C57BL/6 female and male mice were used (Harlan, Indianapolis, IN). Animals were housed in a room with controlled temperature (22 ± 2°C), and a 12:12-hour light-dark cycle (lights on at 6:00 AM) with ad libitum access to
Native FST288 Promotes Slightly Greater Muscle Anabolic Effects than FST315
The first goal was to examine the consequence of local overexpression of the two native FST isoforms (FST288 and FST315) and one engineered isoform that has been relieved of its heparin binding capability (FST288-ΔHBS). Removal of heparin binding activity in FST288 was facilitated by replacing the native HBS found in its FSD1 domain with that of the homologous sequence in follistatin-like 3 as initially described by Sidis et al. (2005). Female C57BL/6 mice were injected with 25 μg of the
Discussion
In this report, the primary objective was to investigate the beneficial application of a novel FST therapeutic in ameliorating the effects of muscle damage in a variety of clinically relevant injury models. We believe that FST’s unique ability in neutralizing multiple members of the TGFβ family results in an improved ability to augment muscle repair more so than merely inhibiting one member alone. Ultimately, we found that FST possesses attributes beyond that of a simple growth factor,
Acknowledgments
The authors thank Bryan Jones, Guilherme Rocha, Rong Wang, Laura Myers, and Jennifer Pereira for the design of gene constructs, histology, statistical analysis, and purification of the proteins.
Authorship Contributions
Participated in research design: Yaden, Dai, Andrews, Krishnan.
Conducted experiments: Yaden, Wang, Shetler, Datta-Mannan, Wilson, Andrews, Milner.
Performed data analysis: Yaden, Wang.
Wrote or contributed to the writing of the manuscript: Yaden, Dai, Datta-Mannan, Croy, Bryant, Andrews, Krishnan
References (50)
et al.
Follistatin complexes Myostatin and antagonises Myostatin-mediated inhibition of myogenesis
Dev Biol
(2004) BilezikjianLM et al.
Autocrine/paracrine regulation of pituitary function by activin, inhibin and follistatin
Mol Cell Endocrinol
(2004) Datta-MannanA et al.
An engineered human follistatin variant: insights into the pharmacokinetic and pharmocodynamic relationships of a novel molecule with broad therapeutic potential
J Pharmacol Exp Ther
(2013) FunabaM et al.
Calcium-regulated expression of activin A in RBL-2H3 mast cells
Cell Signal
(2003) HashimotoO et al.
Difference between follistatin isoforms in the inhibition of activin signalling: activin neutralizing activity of follistatin isoforms is dependent on their affinity for activin
Cell Signal
(2000) HillJJ et al.
The myostatin propeptide and the follistatin-related gene are inhibitory binding proteins of myostatin in normal serum
J Biol Chem
(2002) KocićJ et al.
Interleukin-17 modulates myoblast cell migration by inhibiting urokinase type plasminogen activator expression through p38 mitogen-activated protein kinase
Int J Biochem Cell Biol
(2013) ReameNE et al.
Differential effects of aging on activin A and its binding protein, follistatin, across the menopause transition
Fertil Steril
(2007) SealeP et al.
Pax7 is required for the specification of myogenic satellite cells
Cell
(2000) Sierra-FilardiE et al.
Activin A skews macrophage polarization by promoting a proinflammatory phenotype and inhibiting the acquisition of anti-inflammatory macrophage markers
Blood
(2011)View full text
© 2014 by The American Society for Pharmacology and Experimental Therapeutics