"The mythical fountain of youth has a long history in human imagination, stretching back at least 2,000 years to the writings of Herodotus. In 2005, a seminal manuscript from the laboratory of Tom Rando described an experiment whereby the joining of the circulation of older and younger mice via heterochronic parabiotic pairing resulted in the rejuvenation of stem cells in the older animal (Conboy et al., 2005). The search for the humoral factors affecting the function of aging stem cells was on. Proteomics analysis comparing young and aged serum using SOMAmer technology suggested the TGF-β superfamily member GDF11 as a candidate, since their analysis indicated reduced levels in older mice and presented data that suggested GDF11 was capable of reversing age-related hypertrophy (Loffredo et al., 2013). The authors then reported that systemic injection of GDF11 reverses age-related dysfunction in skeletal muscle (Sinha et al., 2014) as well as vascular and neurogenic function in the brain (Katsimpardi et al., 2014). These findings were surprising because a closely related TGF-β member, myostatin, is a potent inhibitor of skeletal muscle growth (McPherron et al., 1997). Moreover, mice carrying targeted mutations in Gasp-1 and/or Gasp-2, two specific inhibitors of myostatin and GDF11, exhibit impaired muscle regenerative capacity consistent with an upregulation of myostatin and GDF11 signaling (Lee and Lee, 2013). Thus, the mechanism by which GDF11 could improve muscle regeneration and rejuvenate aged satellite cells was not at all clear.

In this issue of Cell Metabolism, Egerman et al. (2015) undertook a careful analysis of the function of GDF11 in young and aged mice. They report the opposite of what Sinha et al. reported, that overexpression of GDF11 results in impaired satellite cell function and reduced muscle regeneration. Notably, systemic delivery of GDF11 into old mice had no effect, whereas in young mice muscle regeneration was delayed due to reduced expansion and differentiation of satellite cells. Myostatin and GDF11 are highly homologous and exhibit 89% identity in amino acid sequence in the mature protein. Egerman and co-workers first assessed the specificity of SOMAmer analysis to identify GDF11. They found that the SOMAmer analysis used in the prior papers (Loffredo et al., 2013 and Sinha et al., 2014) was unable to discriminate between myostatin and GDF11. Similarly, they showed by western blot analysis that the GDF11 antibody previously used by Loffredo et al. (2013) detects both myostatin and GDF11. Notably, this antibody also showed that the combined GDF11/myostatin seems to increase with age, not decrease as previously reported, if the entirety of the signal was accounted for.

To specifically detect GDF11 levels in serum, Egerman et al. established an immunoassay specific for GDF11 protein. As GDF11 levels in mice were below the detection sensitivity for this immunoassay, the authors measured GDF11 concentration in both young and aged rats and humans and demonstrated that GDF11 actually increases during aging. This was further confirmed by RNA-sequencing analysis on rat skeletal muscles from 6 to 24 months of age. Interestingly, while GDF11 mRNA levels increase as a function of age, myostatin mRNA levels undergo a decrease ( Figure 1A).Of particular interest, myostatin and GDF11 share the same receptor and canonical signaling pathway (Trendelenburg et al., 2009). To determine whether GDF11 could have distinct effects on skeletal muscle cells in comparison to myostatin, Egerman et al. treated human primary myoblasts with different doses of GDF11 and myostatin recombinant proteins to decipher their signaling and cellular effects. Importantly, Egerman et al. found that both proteins equivalently activate the canonical SMAD2/3 pathway as well as the myostatin non-canonical MAPK pathways, and similarly inhibit myoblast differentiation (Figure 1B). Microarray analysis did not reveal any significant differences between GDF11- and myostatin-treated myoblasts, supporting the notion that GDF11 does not have a specific function on skeletal muscle cells compared to myostatin. Altogether, these results suggest that GDF11 and myostatin share the same activity on skeletal muscle.

As Sinha et al. reported an improvement of the regenerative ability in aged mice following GDF11 systemic delivery, Egerman et al. sought to re-analyze the function of GDF11 on satellite cell and muscle repair. Daily intraperitoneal injections of GDF11 recombinant protein (rGDF11, 0.1 mg/kg) were performed on 1-year-old mice prior to the cardiotoxin (CTX)-induced muscle injury, as previously described by Sinha et al. At 7 days post-CTX injection, Egerman et al. did not observe any difference in the regenerative capacity of aged skeletal muscle treated with GDF11 or any change in the number of satellite cells.

Interestingly, the administration of a 3-fold-higher dose of GDF11 also failed to improve the regenerative capacity of young skeletal muscle. Worse still, GDF11 delivery dramatically decreases the area of regenerating fibers at 14 days post-CTX injection, suggesting impaired differentiation. The decreased number of myogenin-expressing progenitors on single myofibers treated with GDF11 for 3 days further confirmed that GDF11 delays satellite cell progression and differentiation. Finally, freshly isolated satellite cells from adult and aged mice cultured and treated with GDF11 exhibit a slower rate of proliferation without any significant change in myogenic marker expression, supporting the notion that GDF11 limits satellite cell expansion.

The study of Egerman et al. clearly demonstrates that GDF11 serum levels do not decrease and instead increase during aging; consistent with an increase, their group previously demonstrated that downstream SMAD signaling increases with age (Trendelenburg et al., 2009). They show that systemic injection of GDF11 impairs satellite cell expansion and differentiation, leading to decreased regenerative capacity. This work is entirely consistent with previous studies that identify inhibitory functions of GDF11 on myogenesis and muscle regeneration (Gamer et al., 2001, Lee and Lee, 2013 and Souza et al., 2008). Egerman et al. also demonstrate that GDF11 and myostatin share the same signaling pathways in skeletal muscle cells. This study also underscores the importance of developing therapeutic strategies using inhibitors of GDF11/myostatin signaling to prevent skeletal muscle atrophy and weakness in age-related diseases such as sarcopenia (Lach-Trifilieff et al., 2014).

Clearly, like the mythical fountain of youth, GDF11 is not the long-sought rejuvenation factor. Given the findings of Egerman et al. and the clinical importance of therapeutic strategies involving the inhibition of GDF11/myostatin, the suggested “rejuvenating” activity of GDF11 in the heart and brain (Katsimpardi et al., 2014 and Loffredo et al., 2013) should also be re-examined, since the underlying premise of those other two manuscripts, that GDF11 decreases with age, is contradicted by the Egerman manuscript."