Identifying candidates that rejuvenate aged muscle stem cells is an important strategy toward developing therapies to treat age-related diseases. In this issue,re-investigate the activity of GDF11 in myogenesis, recently suggested as an anti-aging agent, and instead find a potent inhibitory effect on skeletal muscle regeneration.

Main Text

Conboy et al., 2005 Conboy I.M.

Conboy M.J.

Wagers A.J.

Girma E.R.

Weissman I.L.

Rando T.A. 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 (). The search for the humoral factors affecting the function of aging stem cells was on.

Loffredo et al., 2013 Loffredo F.S.

Steinhauser M.L.

Jay S.M.

Gannon J.

Pancoast J.R.

Yalamanchi P.

Sinha M.

Dall’Osso C.

Khong D.

Shadrach J.L.

et al. Sinha et al., 2014 Sinha M.

Jang Y.C.

Oh J.

Khong D.

Wu E.Y.

Manohar R.

Miller C.

Regalado S.G.

Loffredo F.S.

Pancoast J.R.

et al. Katsimpardi et al., 2014 Katsimpardi L.

Litterman N.K.

Schein P.A.

Miller C.M.

Loffredo F.S.

Wojtkiewicz G.R.

Chen J.W.

Lee R.T.

Wagers A.J.

Rubin L.L. McPherron et al., 1997 McPherron A.C.

Lawler A.M.

Lee S.J. Lee and Lee, 2013 Lee Y.S.

Lee S.J. 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 (). The authors then reported that systemic injection of GDF11 reverses age-related dysfunction in skeletal muscle () as well as vascular and neurogenic function in the brain (). These findings were surprising because a closely related TGF-β member, myostatin, is a potent inhibitor of skeletal muscle growth (). 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 (). Thus, the mechanism by which GDF11 could improve muscle regeneration and rejuvenate aged satellite cells was not at all clear.

Egerman et al. (2015) Egerman M.A.

Cadena S.M.

Gilbert J.A.

Meyer A.

Nelson H.N.

Swalley S.E.

Mallozzi C.

Jacobi C.

Jennings L.L.

Clay I.

et al. In this issue of Cell Metabolism,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.

Loffredo et al., 2013 Loffredo F.S.

Steinhauser M.L.

Jay S.M.

Gannon J.

Pancoast J.R.

Yalamanchi P.

Sinha M.

Dall’Osso C.

Khong D.

Shadrach J.L.

et al. Sinha et al., 2014 Sinha M.

Jang Y.C.

Oh J.

Khong D.

Wu E.Y.

Manohar R.

Miller C.

Regalado S.G.

Loffredo F.S.

Pancoast J.R.

et al. Loffredo et al. (2013) Loffredo F.S.

Steinhauser M.L.

Jay S.M.

Gannon J.

Pancoast J.R.

Yalamanchi P.

Sinha M.

Dall’Osso C.

Khong D.

Shadrach J.L.

et al. 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 () was unable to discriminate between myostatin and GDF11. Similarly, they showed by western blot analysis that the GDF11 antibody previously used bydetects 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.

Figure 1 GDF11 and Myostatin Expression from Adulthood to Aging and Their Signaling Pathways Show full caption (A) While myostatin expression decreases from 6 to 24 months in skeletal muscle rat, GDF11 expression increases during this period. (B) Both GDF11 and myostatin bind to the type I and type II receptors, ACTRIIB and ALK-4/5, respectively, to activate both canonical SMAD2/3 and non-canonical MAPK (p38, JNK, ERK1/2) pathways. The myostatin/GDF11 signaling pathways negatively regulate skeletal muscle proliferation and differentiation and protein synthesis, which ultimately results in muscle atrophy in adulthood. ACTRIIB, activin receptor type-IIB; AKT, protein kinase B; ALK4/5, activin-like kinase 4/5; eIF, translation initiation factor; ERK1/2, extracellular signal-regulated kinase 1/2; FOXO, forkhead box protein O; GSK3-β, glycogen synthase kinase 3 β; JNK, c-Jun N-terminal kinase; LC3, microtubule-associated protein 1A/1B-light chain 3; MAFbx, muscle atrophy F-box; MAPK, mitogen-activated protein kinase; mTORC1, mammalian target of rapamycin complex 1; MuRF1, muscle RING-finger protein 1; p70S6K, p70S6 kinase; 4EB-P1, 4E binding protein 1. 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 1 A).

Trendelenburg et al., 2009 Trendelenburg A.U.

Meyer A.

Rohner D.

Boyle J.

Hatakeyama S.

Glass D.J. Of particular interest, myostatin and GDF11 share the same receptor and canonical signaling pathway (). 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 1 B). 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.