Viewpoints
The Immateriality of Circulating GDF11
Buel D. Rodgers
R
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ecent portrayals of growth differentiation factor
(GDF)-11 as an endocrine fountain of youth fail to
consider the competitive influences of myostatin, a homologous protein with true endocrine action that binds
the identical receptor, activin receptor IIb (ActRIIb).
Subsequent studies have disproven the original premise
that circulating levels of GDF11, but not those of myostatin, decline with age as the exact opposite seems to be
true. These latter studies also seriously question the validity of data presented in the previous reports and indicate
that myostatin circulates at levels 500× greater than those
of GDF11; yet, both ligands share nearly identical affinities for ActRIIb. The compromised striated muscle function, neurogenesis, and vascular remodeling that occur
with advanced age are, therefore, not caused by reduced
circulating GDF11 as it could never outcompete myostatin
for shared receptor-binding sites.
yet, the null phenotypes for each ligand differ substantially
with minimal redundancy occurring only in bones.5,6
Two subsequent reports by Egerman et al7 and Smith et
8
al and our own, Rodgers and Eldridge,9 directly refute the
Harvard group’s results and question the validity of reagents
critical to their studies.7–9 In the study by Poggioli et al,10 an
article recently published in this journal, the Harvard group
attempted to address these concerns by collectively assessing GDF11 and myostatin as a single factor “GDF11/8.”10
This conflicts with their foundational claim that reductions
in circulating GDF11, but not myostatin, are responsible for
the age-related decline in striated muscle function, neurogenesis, and vascular remodeling. More confusing is the fact that
Poggioli et al10 assessed the wrong antibody in their critique
of Egerman et al.7
Antibody Cross-Reactivity
Using Western blots without loading controls, Loffredo et al3
reported that plasma GDF11 levels were lower in aged versus
young mice. However, Egerman et al7 demonstrated that the
antibody used in this study (anti-BMP11 recently renamed
anti-BMP11+GDF8/GDF11 antibody, Abcam catalog number, ab124721) cross-reacted with myostatin and that it even
recognized levels of myostatin that were beyond detection for
GDF11. Egerman et al7 further demonstrated nonspecificity
of the GDF11 SOMAmer (Slow-Offrate Modified Aptamers)
used by Loffredo et al3; all of which is hardly surprising as
GDF11 and myostatin are nearly identical homologous proteins that share a common orthologous ancestor.11 Egerman et
al7 then validated a novel GDF11-specific immunoassay using a different antibody (antihuman GDF11; R&D Systems,
clone 743843, catalog number, MAB19581) and determined
that in rats and humans, circulating GDF11 levels rise, not
fall, with age.
Poggioli et al10 published a challenge to Egerman et al7
claiming that the Abcam antibody cross-reacts with circulating immunoglobulin10 despite the fact that Egerman et al7
validated their westerns using recombinant GDF11 as a positive control. Even if Egerman et al7 accidentally quantified
changes in circulating immunoglobulin, these data still do not
refute the immunoassay results with rat and human sera as
they were generated with the R&D Systems mouse monoclonal, not the Abcam rabbit monoclonal. Indeed, this immunoassay was generated specifically because the Abcam antibody
cross-reacted with myostatin. Conclusions by Egerman et
al,7 therefore, were not based on Western blotting data but on
the immunoassay. None of this was discussed in the study by
Poggioli et al,10 which also contained several inaccurate representations of the literature. This includes claims that both
myostatin and GDF11 influence skeletal muscle patterning
when in fact only skeletal patterning is regulated by GDF11
as skeletal muscle size, fiber number, and fiber type are all
Controversial Claims
Demonstrating that a single circulating factor reverses the
effects of aging, particularly in the heart, muscle, and brain,
is beyond transformative as it could revolutionize geriatric
medicine and the treatment of sarcopenia, cancer cachexia,
heart failure, and even stroke. Such a story was recently told.
Unfortunately, however, it is founded on a misunderstanding
of competitive ligand interactions and on questionable science.
With 2 high-profile publications in Science, Katsimpardi
et al1 and Sinha et al,2 and another in Cell, Loffredo et al,3
the same Harvard group suggested that the age-associated
decline in circulating levels of GDF11 can induce cardiac
hypertrophy, skeletal muscle dysfunction, and compromised
neurogenesis.1–3 They also suggested that restoring GDF11 in
aged circulation rejuvenates these tissues and induces vascular
remodeling. The cornerstone of these conclusions is their reported observation that circulating GDF11, but not myostatin
(also known as GDF8), declines with age. This is an extremely
important distinction because both ligands bind the identical
receptor, ActRIIb, with identical picomolar affinities and induce identical signaling pathways.4 Therefore, they should
have identical actions assuming that receptor access is similar;
The opinions expressed in this Viewpoint article are not necessarily
those of the editors or of the American Heart Association.
From the Washington Center for Muscle Biology, Washington State
University, Pullman.
Correspondence to Buel D. Rodgers, PhD, Washington Center for
Muscle Biology, Washington State University, 135 VBR, Pullman, WA
99164. E-mail [email protected]
(Circ Res. 2016;118:1472-1474.
DOI: 10.1161/CIRCRESAHA.116.308478.)
© 2016 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.116.308478
1472
Rodgers The Immateriality of Circulating GDF 1473
Nonstandard Abbreviations and Acronyms
ActRIIb
GDF11
activin receptor IIb
growth differentiation factor 11
unaffected in muscle-specific GDF11 null mice with either
wild-type or myostatin null backgrounds.5
There is no GDF11/8. These are 2 different factors that
regulate different developmental and physiological processes.
The collective assessment of both factors is not only confusing and inaccurate, more importantly, it also ignores the original claim that reduced circulating GDF11 alone is responsible
for the age-associated changes discussed. Furthermore, the
literature discussion by Poggioli et al10 is misleading and this
is particularly true of their representation of our recent study.
Competing Ligands
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In the study by Sinha et al,2 the Harvard group used a sandwich immunoassay (R&D Systems catalog number, DGDF80)
to quantify circulating myostatin in young and aged mice and
although levels in the latter were 20% lower, this difference
was not significant, leading them to conclude that circulating
myostatin levels do not change with age. We used the same
assay and determined that it cross-reacts with GDF11 as the
addition of recombinant GDF11, also from R&D Systems,
produces a false low.9 We demonstrated GDF11 interference
using purified myostatin standards and with serum samples
and have since determined that assay interference occurs even
when samples are acid treated, a protocol requirement that
eliminates interference from follistatin and other myostatinbinding proteins. Most importantly, our study in no way confirms “selective detection of GDF8 (not GDF11)” as Poggioli
et al10 claims because our study suggests that the exact opposite is true.
We also detected serum myostatin immunoreactivity in
myostatin null mice, which cannot be due to myostatin, but
to GDF11. Levels were 500× lower than those in wild-type
mice and are consistent with those reported by Egerman et
al,7 further indicating that the assay itself does not selectively
detect myostatin, but that GDF11 levels are simply too low to
normally cause interference. We then documented a gradual
age-related decline in serum myostatin consistent with the previously reported age-related decline among human subjects.12
Poggioli et al,10 therefore, actually refute the Harvard group’s
previous claims as the reduction in circulating GDF11/8 is due
to changes in myostatin and not in GDF11. In other words,
their new results disprove their original conclusions.
Although 2 commentaries recently summarized this
controversy,13,14 this most salient of points, the fact that circulating GDF11 cannot possibly outcompete circulating
myostatin, was never discussed. Without empirical evidence
suggesting otherwise, we can, therefore, only objectively
conclude that circulating GDF11 is immaterial. All 3 ActRIIb
ligands—GDF11, myostatin, and activin—bind with nearly
identical affinities. This means that receptor occupancy, activation, and biological activity are dependent on ligand
availability, and circulating myostatin is 500× more available
than GDF11. In fact, activin is well known to function as an
autocrine and paracrine factor; yet, it normally circulates at
levels similar or 10-fold higher than those of GDF11.15 By
contrast, several studies suggest that endocrine myostatin attenuates the hepatic growth hormone/IGF1 axis, contributes
to the skeletal muscle atrophy that occurs with heart failure,
and regulates the normal growth and development of adult
skeletal muscle.16–18
Data Analysis Concerns
Studies by the Harvard group presume that what can occur actually does occur. The cardiac actions attributed to GDF11, for
example, could very well be due to myostatin, which inhibits
cardiac hypertrophy and excitation–contraction coupling and
cardiomyocyte growth.19–21 Furthermore, the in vitro studies
by Sinha et al2 do not distinguish ActRIIb ligand–specific
actions especially as many assessments incorporated highly
flawed statistics with type 1 errors.2 This includes pseudoreplication sampling in Figure S3B and inappropriate use of t
tests and Mann–Whitney tests with ≥3 independent variables
(Figures 1–3, S2, S4, S6, S11, S14, and S19). Individual P
values are indicated in their figures and most do not meet the
minimal significance threshold of 0.0083 or 0.0042 when applying a Bonferroni correction, depending on the figure and
panel. This is especially true of Figure S19 where 9 of 10 differences noted lack statistical significance, refuting the claim
that GDF11 stimulates the proliferation of muscle satellite
cells from aged, but not young mice. Sinha et al2 also failed
to consider age and treatment interactions (Figures S13C and
S11A-C) and to account for repeated measures and unequal
sample size (Figure S14). Moreover, it is likely that different
regression analyses (eg, logistic or Poisson) should have been
performed in the many instances where categorical dependent variables (eg, percentages/frequencies and cell counts in
Figures 1, 2, S2, S4, S6, and S8) were influenced by multiple
independent variables and when accounting for measurement
interdependence (Figures 2E and 9A–9C). Finally, Sinha et
al2 seem to have misinterpreted serum lactate and glucose
data by suggesting the coordinated decline in both supports evidence of improved mitochondrial function in aged
rGDF11-treated animals. Serum lactate and glucose are in
general inversely related as lactate is a product of glycolysis
in type II muscle fibers, whereas mitochondrial activity reflects anaerobic metabolism in type I fibers. Furthermore, mitochondrial dysfunction is well known to increase lactate and
reduce glucose levels in blood because of a greater reliance of
glycolysis in type II fibers. Thus, the lactate and glucose data
do not support the claimed enhancement of mitochondrial
function by GDF11 administration (Figure S13).
The flawed statistical design and other serious problems
question the reliability of the Sinha et al study.2 Moreover, the
demonstration that circulating myostatin levels are demonstrably higher than those of GDF11 and that levels of the former,
but not the latter, decline with age refutes the fundamental
claim that declining GDF11 compromises striated muscle
function, neurogenesis, and vascular remodeling. Katsimpardi
et al,1 Loffredo et al,1,3 and Sinha et al2 are, therefore, highly
suspect. The same can also be said of Poggioli et al10 and its
erroneous critique of Egerman et al7 and misleading use of
1474 Circulation Research May 13, 2016
“GDF11/8.” Autocrine or paracrine GDF11 or even activin
may in fact regulate some of these processes as the functional
significance of any particular ActRIIb ligand is dependent on
its competitive availability. In aging animals, however, circulating myostatin, not GDF11 and especially not GDF11/8, is
the relevant hormone.
Sources of Funding
B.D. Rodgers was supported by grants from the National Science
Foundation (1147275), the National Institutes of Health (HD067449
to M. Du and B.D. Rodgers), and the LGMD2iFund.
Disclosures
None.
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Key Words: aging ■ cardiomegaly
■ ligands ■ myostatin
■
growth/differentiating factor 11
The Immateriality of Circulating GDF11
Buel D. Rodgers
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Circ Res. 2016;118:1472-1474
doi: 10.1161/CIRCRESAHA.116.308478
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