J Appl Physiol 129: 272–282, 2020.

First published July 9, 2020; doi:10.1152/japplphysiol.00381.2020.

REVIEW Edward F. Adolph Distinguished Lecture

Edward F. Adolph Distinguished Lecture. Skeletal muscle atrophy: Multiple

pathways leading to a common outcome
Sue C. Bodine
Department of Internal Medicine/Endocrinology and Metabolism, University of Iowa Carver College of Medicine, Iowa City,
Iowa
Submitted 15 May 2020; accepted in final form 3 July 2020

Bodine SC. Edward F. Adolph Distinguished Lecture. Skeletal muscle atrophy:

multiple pathways leading to a common outcome. J Appl Physiol 129: 272–282,
2020. First published July 9, 2020; doi:10.1152/japplphysiol.00381.2020.—Skele-
tal muscle atrophy continues to be a serious consequence of many diseases and
conditions for which there is no treatment. Our understanding of the mechanisms
regulating skeletal muscle mass has improved considerably over the past two
decades. For many years it was known that skeletal muscle atrophy resulted from
an imbalance between protein synthesis and protein breakdown, with the net
balance shifting toward protein breakdown. However, the molecular and cellular
mechanisms underlying the increased breakdown of myofibrils was unknown. Over
the past two decades, numerous reports have identified novel genes and signaling
pathways that are upregulated and activated in response to stimuli such as disuse,
inflammation, metabolic stress, starvation and others that induce muscle atrophy.
This review summarizes the discovery efforts performed in the identification of
several pathways involved in the regulation of skeletal muscle mass: the mamma-
lian target of rapamycin (mTORC1) and the ubiquitin proteasome pathway and the
E3 ligases, MuRF1 and MAFbx. While muscle atrophy is a common outcome of
many diseases, it is doubtful that a single gene or pathway initiates or mediates the
breakdown of myofibrils. Interestingly, however, is the observation that upregula-
tion of the E3 ligases, MuRF1 and MAFbx, is a common feature of many divergent
atrophy conditions. The challenge for the field of muscle biology is to understand
how all of the various molecules, transcription factors, and signaling pathways
interact to produce muscle atrophy and to identify the critical factors for interven-
tion.
MAFbx; mTORC1; MuRF1; protein synthesis; ubiquitin proteasome pathway

INTRODUCTION signals from multiple systems (neural, endocrine, cardiovascu-

lar) and organs (liver, adipose, gut) within the body compli-
Skeletal muscle is a complex and dynamic tissue that com-
cates the efforts to identify the key regulators of skeletal
prises ~40% of body weight and performs critical functions
muscle mass.
related to movement (power output and sensory feedback),
Muscle size is a very plastic characteristic of limb muscles,
metabolism (substrate utilization, storage, and supply) and
changing over the life span with different signals playing
thermogenesis. Skeletal muscle mass has been shown to be
critical regulatory roles at each life stage (Fig. 1). Alterations
predictive of longevity in older adults and is a critical variable
in muscle mass can occur as a consequence of changes in both
in predicting mortality as a consequence of diseases such as
the number and/or size of individual muscle fibers depending
cancer, type II diabetes, and cardiovascular disease (75, 80).
on the life stage. The number of fibers in a muscle is estab-
The regulation of skeletal muscle mass is multifactorial inte-
lished during embryonic and fetal development and is depen-
grating signals from hormones, growth factors, cytokines,
dent on the proliferation, differentiation and fusion of myo-
nutrients, load and activity to regulate multiple intersecting
blasts to form myofibers (37, 51). The number of muscle fibers
pathways that control the balance between protein synthesis
in a muscle remains constant in healthy muscles throughout
and protein degradation (63). Furthermore, skeletal muscle is a
most of the life span, with decreases occurring during ad-
multicellular tissue that relies on an intact vascular system and
vanced aging (51). In contrast, changes in fiber size can occur
motor/sensory innervation to maintain its size and function.
throughout the life span. Increases in both the length and
The fact that skeletal muscle interacts with and integrates
cross-sectional area of individual fibers occur before and dur-
ing puberty and are dependent on satellite cell proliferation and
Correspondence: S. C. Bodine (Sue-Bodine@uiowa.edu). differentiation (67, 82). The predominant change that occurs
272 8750-7587/20 Copyright © 2020 the American Physiological Society http://www.jap.org
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 REGULATION OF SKELETAL MUSCLE ATROPHY
postpuberty is to fiber cross-sectional area, resulting in both
increases (i.e., hypertrophy) and decreases (i.e., atrophy) in
size. The role of satellite cells in adaptive hypertrophy of adult
muscle is still debated, but recent evidence suggests that
adaptive muscle hypertrophy can occur without satellite cells
and the addition of myonuclei to individual muscle fibers (9,
24, 54).
Our understanding of the factors and pathways that regulate
both hypertrophy and atrophy of skeletal muscle has increased
greatly over the past two decades. This review is a summary of
the Adolph Distinguished Lecture given by me at the 2019
Experimental Biology meeting. The work presented in this
review reflects a journey that started for me as a graduate
student interested in understanding skeletal muscle plasticity
and in particular how force is generated and regulated in
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273
Fig. 1. Regulation of skeletal muscle mass
throughout the life span. This diagram illus-
trates some of the critical factors that regu-
late skeletal muscle size at different life
stages. At each life stage skeletal muscle
undergoes specific changes that are regu-
lated by different critical factors. During the
embryonic stage growth factors, hormones
and myocytes/satellite cells play a critical
role in the formation and innervation of
muscle fibers. During the postnatal stage,
external loading and increased neural activa-
tion become important factors along with
growth factors, hormones, and satellite cells
in regulating increases in the length and
cross-sectional area of muscle fibers. As
adults, maintaining muscle mass and adapt-
ing to changes in external loading and neural
activation become the dominant focus. With
advancing age, decreases in external loading
and activity, as well as, inflammation, in-
creasing levels of cytokines, oxidative stress,
and metabolic stress can lead to a decrease in
muscle mass and strength.
skeletal muscle to control movement. Over the course of my
career my research became more focused on undercovering the
mechanisms regulating skeletal muscle size, especially under
conditions leading to the loss of muscle mass and function. In
this review, I will highlight research conducted since the late
1990s that has contributed to a greater understanding of the
mechanisms underlying skeletal muscle atrophy.
In the 1990s our understanding of the cellular and molecular
mechanisms regulating skeletal muscle was relatively limited.
It was known that loss of muscle mass occurred under a variety
of diseases and conditions such as immobilization, spinal cord
injury, aging and others as shown in Fig. 2. In animal models,
muscle atrophy could be induced by decreases in neural activ-
ity, decreases in external loading, increases in glucocorticoids,
increases in inflammatory cytokines and nutrient deficiency
Fig. 2. Skeletal muscle atrophy is prevalent
in many diseases. Skeletal muscle atrophy is
a serious consequence of the diseases and
conditions listed in this diagram.
274 REGULATION OF SKELETAL MUSCLE ATROPHY

(30, 52, 66). The induction of muscle atrophy was related to an
imbalance between protein synthesis and degradation with the
net balance being shifted toward protein breakdown. There
were data to suggest that both decreases in protein synthesis
and increases in protein degradation contributed to muscle loss,
and that the relative contribution of each process to muscle loss
depended on the conditions (3, 28, 29, 43, 56, 62); however,
unknown at the time was the identity of the signaling pathways
responsible for the changes in protein synthesis and degrada-
tion.
In the late 1990s, while at Regeneron Pharmaceuticals, we
initiated a discovery program aimed at determining the signal-
ing pathways regulating the loss of skeletal muscle mass. To
approach this complex problem, we developed a multi-disci-
plinary muscle research program diagrammed in Fig. 3. At the
center of the program was the establishment of rodent models
of muscle atrophy and hypertrophy, from which skeletal mus-
cles were obtained at multiple time points postexperimental
manipulation and then used for the identification of signaling
pathways and differentially regulated genes and proteins. A
variety of pharmacological agents were also used to induce
atrophy (e.g., dexamethasone, cytokines) and hypertrophy
(e.g., clenbuterol) or block specific enzymes and signaling
pathways (e.g., rapamycin). Finally, emerging technologies,
such as mouse genetic engineering and in vivo electroporation,
were utilized to overexpress or delete specific genes in skeletal
muscle. These approaches allowed for the discovery of novel
pathways involved in the regulation of skeletal muscle mass.
In the following sections, a summary of work that led to the
discovery of novel signaling pathways involved in the regula-
tion of skeletal muscle mass is presented. It begins with a
description of the experiments that identified mTORC1 activa-
tion as a critical regulator of skeletal muscle hypertrophy in
adult mammals; and continues with a description of the exper-
imental approaches taken to identify novel signaling pathways
involved in the loss of skeletal muscle mass. Details are
provided regarding the discovery of two skeletal muscle-
specific E3 ubiquitin ligases, MuRF1 and MAFbx/Atrogin1,
followed by highlights of published findings related to the
transcriptional regulation of MuRF1 and MAFbx and their
expression patterns during different forms of atrophy, as well
as, the use of mice with global deletions of either MuRF1 or
MAFbx to identify the potential mechanisms of action of these
E3 ligase in the regulation of skeletal muscle mass. The final
sections discuss the link between MuRF1 and MAFbx expres-
sion and proteasome activity, and the continuing search for
MuRF1 and MAFbx substrates. The review concludes by
highlighting some of the important questions that remain to be
answered.
THE IDENTIFICATION OF MTORC1 AS A MAJOR
REGULATOR OF SKELETAL MUSCLE SIZE IN MAMMALS
Skeletal muscle is highly responsive to changes in external
loading, increasing in size in response to enhanced loading and
decreasing in size in response to reduced loading (66). It had
been shown in both human and rodent muscle, that the rate of
protein synthesis is modified in response to changes in external
loading. Following an acute bout of resistance exercise in
humans (87) or simulated resistance exercise in rats (84),
protein synthesis increases; whereas, the early response to
conditions that decrease loading, such as immobilization or
bedrest, is a decrease in protein synthesis (25, 28). While
robust changes in protein synthesis in response to alterations in
loading had been demonstrated, the cellular mechanisms re-
sponsible for the changes were unknown. Some possible can-
didates at the time included Insulin-like Growth Factor 1
(IGF-1), calcineurin, and the PI(3)K/Akt signaling pathway.
The case for IGF-1 as a major mediator of growth and protein
synthesis in skeletal muscle was supported by reports that
IGF-1 induces an increase in myotube size in vitro (65, 77),
IGF-1 is elevated in vivo following acute high-load contrac-

Fig. 3. Building a multidisciplinary muscle re-

search program. A multidisciplinary research
program was developed to discover novel mech-
anisms regulating skeletal muscle size. At the
center of the program was the establishment of
animal models of skeletal muscle atrophy (de-
nervation, nerve crush-reinnervation, joint im-
mobilization, hindlimb suspension, glucocorti-
coid excess) and hypertrophy (functional over-
load, reloading following disuse). Muscle
tissues taken at different time points were used
in the identification of novel gene and protein
targets using differential gene expression and
proteomics approaches. Muscle samples were
also used to identify signaling pathways that
were altered in response to stimuli that induced
atrophy and hypertrophy. Furthermore, pharma-
cological agents, such as clenbuterol, IGF1,
rapamycin, glucocorticoids, were utilized both
in vivo and in vitro to manipulate selective
pathways to induce changes in muscle size.
Finally, technologies such as in vivo electropo-
ration and mouse genetic engineering were uti-
lized to modify gene expression in skeletal
muscle.

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 REGULATION OF SKELETAL MUSCLE ATROPHY
tions (1), and transgenic overexpression of IGF-1 in mice
results in enhanced muscle size (2, 18). Another pathway that
had been linked to growth in model organisms and mice was
the PI(3)K/Akt/mTOR pathway. Genetic manipulation in Dro-
sophila of PI(3)K, PKB/Akt and S6K1 (p70s6k) revealed that
deletion of components of this pathway resulted in smaller
cells, but not fewer cells, suggesting that the PI(3)K/Akt/
mTOR pathway played a major role in the regulation of cell
size (57, 81). Further support for this pathway came from the
deletion of S6K1 in mice which led to lower body weights and
organ growth relative to wild-type (WT) littermates (72).
Finally, data were emerging that the PI(3)K/Akt/mTOR path-
way may play a role in skeletal muscle growth in response to
resistance exercise. Baar and Esser demonstrated that follow-
ing high resistance exercise training in rats, phosphorylation of
S6K1 (p70s6k) increased in those muscles experiencing an
increase in loading resulting in hypertrophy (4). Given that
S6K1 is downstream of mTORC1, these data suggested that
activation of mTORC1 and its downstream targets were im-
portant for muscle growth.
Given what was known at the time, we asked the following
questions: Is the PI(3)k/Akt/mTOR pathway regulated in vivo
during hypertrophy or atrophy, and what happens to growth or
atrophy when this pathway is inhibited? To address the first
question, we used synergist ablation, a model of functional
overload, to determine whether components of the PI(3)k/Akt/
mTORC1 pathways were activated in a muscle undergoing
hypertrophy. We found that following synergist ablation, all
components of the PI3k/Akt/mTORC1 were activated in the
plantaris muscle and that activation of this pathway was an
early response (12). Phosphorylation and total amount of
Akt/PKB increased early leading to activation of mTORC1 as
measured by (1) an increase in phosphorylation of mTORC1 at
Ser2448 (64), (2) an increase in the phosphorylation and
activity of S6K1/p70s6k (12), and (3) a decrease in the amount
of 4EBP1/PHAS-I bound to eIF4E coupled with an increase in
the amount of eIF4E bound to eIF4G (64). These data were the
first to show that activation of the Akt/mTORC1 pathway was
associated with skeletal muscle growth in vivo. Subsequent
studies have shown that this pathway is activated in human
muscle in response to resistance exercise and associated with
an increase in protein synthesis (21, 22, 34, 58). The question
remained as to whether activation of this pathway was neces-
sary for skeletal muscle growth. To address this question, adult
rats were given rapamycin to inhibit mTORC1 and its down-
stream targets, without affecting activation of Akt or inhibition
of GSK3. Rapamycin treatment for a duration of up to 14 days
prevented 95% of hypertrophy of both slow and fast fibers in
the rat plantaris muscle following functional overload (12),
revealing that activation of mTORC1 was necessary to achieve
adaptive hypertrophy of adult skeletal muscle. Activation of
the Akt/mTORC1 pathways was also shown to decrease in
response to hindlimb unloading, which induced atrophy, and to
increase in response to reloading, which stimulated muscle
regrowth (12). Interestingly, rapamycin treatment given upon
return of weight-bearing locomotion following hindlimb un-
loading significantly inhibited growth of the soleus, plantaris
and medial gastrocnemius muscles; however, the inhibition
was not as great as that seen in the functional overload model.
These data suggest that muscle regrowth following a period of
atrophy is not identical to muscle growth in response to
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275
increased loading associated with resistance exercise in healthy
muscle. The differences between the two growth models, i.e.,
functional overload and unloading/reloading, have not been
explored in detail. The increase in Akt/mTORC1 activation in
the functional overload model was presumed to be in response
to an increase in IGF-1 signaling since IGF-1 had been shown
to activate the PI3k/Akt/mTORC1 pathway in myotubes and
induce hypertrophy (65), and transgenic overexpression of
IGF1 in embryonic mice resulted in larger muscles (18).
However, a subsequent study by Spangenburg et al. (74)
revealed that activation of the IGF1 receptor was not required
for the phosphorylation of Akt and S6K1/p70S6K and the
induction of muscle hypertrophy in response to functional
overload.
In summary, many studies have now shown that activation
of mTORC1 and increases in protein synthesis are critical for
achieving muscle hypertrophy in response to increased loading
in adult mammals. This increase in protein synthesis and
stimulation of muscle fiber growth does not appear to be
dependent on an increase in satellite cell proliferation and the
addition of myonuclei to the muscle fiber (40, 54). Further-
more, suppression of mTORC1 and decreased protein synthe-
sis can contribute to the loss of muscle mass, especially under
conditions of disuse. These results might suggest that activat-
ing the mTORC1 pathway would be a good strategy for
treating muscle atrophy. In fact, it has been shown that over-
expression of Akt in muscle can lead to hypertrophy and
prevent muscle atrophy (12, 49). However, chronic activation
of mTORC1 has been shown to be deleterious to muscle,
leading to muscle atrophy (35). Closer inspection of how
mTORC1 is activated in muscle shows that in resting adult
muscle mTORC1 is at a relatively low activation state. Acti-
vation of mTORC1 occurs periodically as the result of in-
creased loading or amino acid ingestion (19, 33). In healthy
adult muscle, mTORC1 is rarely in a chronically activated
state, and short-term rapamycin treatment in adult animals does
not affect muscle mass (12). Furthermore, a recent study
showed that deletion of raptor and suppression of mTORC1
activity in resting adult mice for 5 mo had no effect on muscle
mass (36). An exception is muscle in aged animals where
mTORC1 activation is chronically elevated under resting con-
ditions and shows reduced activation in response to anabolic
stimuli. Chronic activation of mTORC1 is thought to contrib-
ute to the loss of mass and function with age, and a recent study
suggests that partial suppression of mTORC1 in older animals
could be beneficial (41). It should be noted that mTORC1 is
also activated in muscle upon denervation, a condition that
causes muscle atrophy (29). Interestingly, inhibition of
mTORC1 with rapamycin during denervation results in an
attenuation of muscle atrophy (44). The mechanisms underly-
ing the beneficial effects of rapamycin during denervation and
possibly aging remain unclear and require further investiga-
tion.
IDENTIFICATION OF MUSCLE ATROPHY PATHWAY
The loss of muscle mass occurs as the result of many
conditions and diseases, as shown in Fig. 2. To identify novel
pathways responsible for muscle atrophy, we initially focused
our discovery efforts on models of disuse atrophy. Disuse
atrophy occurs as the result of decreases in external loading, as
276 REGULATION OF SKELETAL MUSCLE ATROPHY

well as, decreases in neural activation of muscle, and ranges in
the degree of muscle loss; being less severe in bedrest (mod-
eled as hindlimb suspension in animals) and most severe
following denervation (Fig. 4). Examination of the extent of
atrophy (% change in muscle mass) of the rat medial gastroc-
nemius over 14 days in three models of disuse (hindlimb
unloading, ankle joint immobilization, denervation) shows that
the rate of loss over the first 3 days was similar across the three
models, whereas after 7 days the rate of loss diverges across
the models (Fig. 4). In an effort to discover potential early
triggers of muscle atrophy, a differential expression analysis
(GeneTag method) was performed on control and 3-day im-
mobilized rat medial gastrocnemius muscle (11) (Fig. 4). In the
primary screen we identified genes that were differentially
regulated by threefold in the immobilized muscle. A secondary
screen was then performed to identify the subset of genes that
were also differentially regulated following denervation and
hindlimb suspension, in addition to immobilization. The sec-
ondary screen identified two genes that were upregulated in all
disuse atrophy models tested, as well as, dexamethasone and
interleukin-1 induced atrophy. Both genes were shown to be
E3 ubiquitin ligases: one gene was a RING finger protein
previously identified in the heart as MURF1 (Trim63) and the
other gene was a novel protein that contained an F-box domain
and was shown to be a member of the Skp-Cullin1-F-box
(SCF) protein family of E3 ubiquitin ligases. We named this
novel protein MAFbx for Muscle Atrophy F-box (FBX032).
This same protein was also identified by Goldberg and col-
leagues in the mouse gastrocnemius muscle following starva-
tion using an Affymetrix microarray and called Atrogin-1 (32).
Since their initial identification in 2001, my laboratory has
been studying the role of MuRF1 and MAFbx in the regulation
of skeletal muscle mass by examining their expression patterns
under a variety of atrophy and hypertrophy conditions, their
transcriptional regulation, and the impact of their deletion on
the loss of muscle mass in different atrophy conditions.
MuRF1 and MAFbx Are Excellent Markers of Muscle
Atrophy
MuRF1 and MAFbx demonstrate many characteristics that
make them excellent markers of muscle atrophy. First, both
genes are selectively expressed in muscle tissue (skeletal,
cardiac and smooth muscle), and in resting skeletal muscle
these genes are expressed at relatively low levels (11). Second,
the expression of both genes rapidly increases in response to a
variety of stressors including unloading, decreased neural ac-
tivity, elevated glucocorticoids, elevated cytokines, increased
oxidative stress, and malnutrition (10, 11, 32, 61). The expres-
sion patterns of both genes vary depending on the atrophy
stimulus. For example, in response to unloading and denerva-
tion, expression of both MuRF1 and MAFbx increases rapidly
within 48 h reaching a peak around 7–10 days and then
gradually declining to baseline by 14 days. Recently, it was
confirmed using mass spectrometry that MuRF1 protein ex-
pression in mouse muscle changed over the same time course
as the mRNA following denervation (50). Validation of protein
expression, especially of MuRF1, has been difficult because of
the lack of specificity of commercially available antibodies.
We have tested the majority of commercially available MuRF1
antibodies, and found positive staining with the MuRF1 anti-
body when used on lysates from muscles taken from MuRF1
KO mice. Moreover, in these KO tissues we often observe a
positive band at the same molecular weight as predicted for
MuRF1 (8). Our data suggest that the majority of commercial

Fig. 4. Discovery of MuRF1 and MAFbx in skeletal muscle. A: animal models of disuse atrophy range in severity from those that primarily produce decreased
external loading of the muscles, such as hindlimb unloading to those that produce both a decrease in external loading and a complete absence of neural activity
such as denervation or spinal cord injury. Joint immobilization is a model that produces decreased external loading and a reduction in neural activity depending
on the degree to which the joint is immobilized. B: loss of mass of the rat medial gastrocnemius muscle (MG) was compared in three models of disuse atrophy
(hindlimb suspension, ankle joint immobilization, and denervation). To identify a potential common trigger of muscle atrophy, 3 days postimmobilization was
chosen as the time point and model to perform a differential gene expression analysis (GeneTag, Applied Bioscience). In the primary screen, all those genes that
were differentially regulated 3-fold were identified. A secondary screen was utilized to identify those genes that were similarly regulated in three disuse models
(immobilization, hindlimb unloading, and denervation). The secondary screen consisted of using Northern blots to analyze gene expression in the medial
gastrocnemius muscle over a time course of atrophy (0, 1, 3, 7 days) for each of the disuse models. From the secondary analysis, two genes (MuRF1 and MAFbx)
were identified that were similarly upregulated in all atrophy models examined.

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 REGULATION OF SKELETAL MUSCLE ATROPHY
antibodies for MuRF1 are nonspecific and should not be used
to quantify MuRF1 protein expression in muscle tissue.
The expression patterns of both MuRF1 and MAFbx in
response to elevated glucocorticoids and cytokines differ com-
pared with disuse; rising rapidly to a peak within days and then
maintaining an elevated level for as long as the signal (gluco-
corticoid or cytokine levels) is present (5). Interestingly, under
increased loading conditions (such as reloading following un-
loading, reinnervation following nerve injury, and functional
overload) the expression of MuRF1 and MAFbx is suppressed
below baseline (6, 7). While MuRF1 and MAFbx expression
are suppressed at 7 days following functional overload, the
expression of both genes is transiently increased at 1 day
following the synergist ablation surgery (6). This same pattern
of elevated expression is seen immediately following an acute
bout of intensive eccentric contractions in humans (53, 86).
The mechanism underlying the suppression of these genes in
response to increased loading is unknown. One possible mech-
anism could be a change in the redox state of the muscle upon
loading. In summary, MuRF1 and MAFbx are excellent mark-
ers of muscle atrophy due to the fact that in all atrophy models
tested to date expression of MuRF1 and MAFbx has been
shown to increase at some time point during the course of the
disease.
MuRF1 and MAFbx Are Regulated By Multiple
Transcription Factors
Examination of the expression patterns of MuRF1 and
MAFbx under different atrophy conditions has revealed two
interesting observations: 1) both genes are upregulated together
under most atrophy conditions and 2) both genes are strongly
induced by the synthetic glucocorticoid, dexamethasone. Based
on these observations, we were interested in understanding the
transcriptional regulation of these genes under different atro-
phy conditions. We started our investigation into the transcrip-
tional regulation of MuRF1 and MAFbx by first examining the
proximal promoter regions, 5,000 bp upstream of the transcrip-
tional start site, of both genes for putative transcription factor
binding sites. Interestingly, we found that the proximal region
of the MuRF1 promoter, but not the MAFbx promoter, con-
tained a perfect palindromic glucocorticoid response element
(GRE) (78). We also found that the proximal promoter region
of each gene contained several consensus Class O forkhead
(FOXO) binding sites (FBE), capable of binding FOXO1,
FOXO3a, and FOXO4; all of which are expressed in skeletal
muscle. This finding was particularly interesting since several
reports had shown that activated FOXO1 (76) could increase
MuRF1 transcription in myotubes, while activated FOXO3a
could increase MAFbx expression in vitro and in vivo (68).
Our studies revealed for the first time that dexamethasone
could directly activate the MuRF1 promoter, but not the
MAFbx promoter (78). Furthermore, we provided additional
evidence for direct activation of the MuRF1 and MAFbx
promoters by the FOXO transcription factors. Our studies,
however, revealed that while the promoters of both MuRF1
and MAFbx could be activated by all of the FOXO transcrip-
tions factors, MAFbx was much more responsive than MuRF1.
Moreover, the promoters, were not equally activated by all of
the FOXO transcription factors. For example, activation of the
MAFbx promoter was greatest in response to FOXO3a and
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277
least in response to FOXO1. In contrast, the MuRF1 promoter
was similarly activated by FOXO1 and FOXO3a. Interestingly,
we found a synergistic activation of the MuRF1 promoter only
by FOXO1 and the activated glucocorticoid receptor (78). The
differential response of MuRF1 and MAFbx to the various
FOXO transcription factors is important because many studies
treat all of the FOXO transcription factors as interchangeable.
FOXO3a and FOXO1 are the predominate FOXO transcription
expressed in skeletal muscle. Many published reports utilize
either FOXO1 or FOXO3a in their studies, and then assume
that the results apply to all FOXO transcriptional factors. A
study by Milan et al. illustrates the differential gene regulation
of MuRF1 and MAFbx in vivo by FOXO1 and FOXO3a (55).
In their study, Milan et al. measured expression of MuRF1 and
MAFbx following denervation in WT mice and mice with a
muscle-specific deletion of FOXO1 or FOXO1/3a/4. In re-
sponse to denervation, the expression of both FOXO1 and
FOXO3a increases, as does the expression of MuRF1 and
MAFbx. Deletion of only FOXO1a in muscle significantly
suppressed the activation of MuRF1, but not MAFbx. In
contrast, deletion of all three FOXOs in muscle resulted in a
significant suppression of both MuRF1 and MAFbx. Interest-
ingly, MuRF1 and MAFbx still increased in response to de-
nervation even in the absence of the FOXO transcription
factors. This activation is likely not related to an activated
glucocorticoid receptor since we showed that upregulation of
both MuRF1 and MAFbx following denervation was similar in
WT and muscle-specific GR knockout mice (79). These data
do illustrate that transcriptional control of MuRF1 and MAFbx
is complex, and the literature shows that multiple transcription
factors can activate both genes. In addition to the glucocor-
ticoid receptor and FOXO1/FOXO3a, the following tran-
scription factors have been shown to activate MuRF1 and
MAFbx: NF-␬B (15, 39, 85), KLF15 (73), C/EBP ␤ (88, 89)
and Smad3 (13).
Deletion of MuRF1, but not MAFbx, Results in Sparing of
Muscle Mass
The substrates targeted for ubiquitination by MuRF1 and
MAFbx remain poorly defined, and thus the mechanisms by
which upregulation of MuRF1 and/or MAFbx contribute to
muscle atrophy remain poorly understood. The generation of
mice with a global deletion of either MuRF1 or MAFbx has
assisted us in understanding the role of these E3 ligases in the
regulation of skeletal muscle mass (11). The phenotype of the
knockout mice has been previously described (10). In brief,
both knockout strains develop normally and show no pheno-
type until later in life (~16 –18 mo) when the MAFbx KO mice
die prematurely of congestive heart failure. The MuRF1 KO
mice have a normal life span and have preserved muscle mass,
but not function, with age (38). Muscle atrophy has been
examined in both knockout (KO) strains and the results have
revealed that, in general, deletion of MuRF1 leads to better
functional muscle sparing than deletion of MAFbx. Deletion of
MuRF1 leads to functional sparing of muscle mass following
denervation (31), hindlimb unloading (48), exogenous gluco-
corticoid treatment (5), and acute lung injury (26). In contrast,
deletion of MAFbx only spares muscle mass following dener-
vation, however, the sparing is not functional. Histological
analysis of denervated muscle from MAFbx KO mice after 14
278 REGULATION OF SKELETAL MUSCLE ATROPHY
days reveals an increase in vacuole-containing fibers and an
increase in fibrosis (31). Interestingly, muscle sparing in the
MuRF1 KO mice following denervation occurs even though
MAFbx expression is higher than measured in WT denervated
muscle and is sustained at a higher level for a longer period of
time as compared with WT mice (31). In general, our muscle
atrophy studies in the KO mice have revealed that suppression
of MAFbx is not required for sparing of muscle mass, however,
suppression of MuRF1 is necessary to achieve muscle sparing.
The one atrophy model where muscle atrophy is not affected
by deletion of either MuRF1 or MAFbx is nutritional depriva-
tion (5).
Increases in MuRF1 and MAFbx Expression are not Always
Linked to Increases in Proteasome Activity
MuRF1 and MAFbx are E3 ubiquitin ligases that are ex-
pressed at low levels in resting muscle and increase rapidly in
response to stimuli that induce muscle atrophy. Given these
facts, it has been hypothesized that increases in MuRF1 and
MAFbx expression lead to muscle atrophy by increasing pro-
teasome activity and protein degradation. In our atrophy mod-
els, we have examined the relationship between the expression
levels of MuRF1 and MAFbx and proteasome activity. Protea-
some activity is measured using in vitro assays in which the
activity of the specific proteasome subunits (␤1, ␤2, and ␤5) is
determined using specific proteasome inhibitors and substrates
in the presence (26S proteasome) or absence (20S proteasome)
of ATP (31). Following denervation, the expression of both
MuRF1 and MAFbx significantly increases by 3 days and then
decreases to baseline by 14 days. In comparison, at 3 days of
denervation, proteasome activity is elevated for only a couple
of subunits (26S ␤1 and ␤2), whereas, at 14 days proteasome
activity is significantly elevated in all subunits (26S (␤1, ␤2,
␤5, and 20S ␤1, ␤2, ␤5) (31). In MuRF1 KO mice, at 3 days
of denervation, proteasome activity is elevated in the 26S ␤2
subunit only, while at 14 days of denervation, proteasome
activity is significantly elevated in all subunits. Interestingly,
the increase in proteasome subunit activity is significantly
higher in the KO mice compared with WT mice. The finding
that proteasome activity increased to a greater extent in the
MuRF1 KO mice than the WT mice was unexpected since
higher proteasome activity has generally been linked to an
increase in muscle atrophy (56), and a report by Cohen et al.
suggested that proteasome activity was decreased in the
MuRF1 KO mice (16).
An elevation in proteasome activity in the MuRF1 KO mice
was also found in response to aging. Examination of old (24
mo) WT and MuRF1 KO male mice revealed that muscle mass
was maintained in the MuRF1 KO mice with age, while it
decreased in WT mice (38). Measurement of proteasome
activity revealed a decrease in the activity of all of the protea-
some subunits as a consequence of aging in WT mice. In
contrast, old MuRF1 KO mice had higher proteasome subunit
activities compared with WT. These data suggest that an
elevated proteasome activity is protective under some condi-
tions, contributing to an increase in protein quality control and
a decrease in cellular stress. The mechanism(s) by which a
deletion of MuRF1 leads to an increase in proteasome activity
are unclear and are under study.
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The use of MuRF1 and MAFbx expression as markers of
increased proteasome activity, and by extension protein deg-
radation, is widespread in the literature. As mentioned previ-
ously, MuRF1 and MAFbx expression are excellent markers of
muscle atrophy; however, an increase in their expression levels
does not always coincide with an increase in proteasome
activity. In the case of denervation, the increase in proteasome
activity occurs as the expression levels of MuRF1 and MAFbx
are decreasing back to baseline. In another disuse model,
hindlimb suspension, expression of MuRF1 and MAFbx in-
creases in the soleus, medial gastrocnemius and tibialis anterior
muscles with unloading, peaking at around 7 days and return-
ing to baseline by 14 days (8). In comparison, proteasome
activity increases only in the soleus and rises continuously over
14 days of unloading. Another example of the disconnect
between MuRF1 and MAFbx expression and proteasome ac-
tivity is functional overload. In response to functional over-
load, MuRF1 and MAFbx expression significantly increases at
day 1 following the synergist ablation surgery and decreases to
baseline by 3 days followed by suppression below baseline (6).
In contrast, proteasome activity increases immediately follow-
ing surgery and continues to rise until around day 7 post
functional overload, returning to baseline levels by 14 days.
These data highlight the fact that elevated proteasome activity
is not always a sign of atrophy, but is also required for
remodeling and growth. These data also strongly suggest that
MuRF1 and MAFbx expression should not be used as substi-
tute markers for proteasome activity or protein degradation.
Identification of MuRF1 and MAFbx Substrates
MuRF1 and MAFbx were first identified as atrophy-associ-
ated E3 ligases expressed selectively in muscle in 2001 (11,
32), however, much is still unknown regarding how their
upregulation contributes to the loss of skeletal muscle mass.
We have compared the response of WT and MuRF1 KO mice
to denervation and dexamethasone treatment and found that
while muscle mass is spared in the KO mice under both
conditions, the mechanisms of action appear to be very differ-
ent (5, 31). For example, following dexamethasone treatment,
muscle sparing in the MuRF1 KO mice appears to be related to
the suppression of FOXO1 gene expression and the mainte-
nance of protein synthesis (5). In comparison, following de-
nervation, muscle sparing in the MuRF1 KO mice is related to
the suppression of genes associated with inactivity (such as
HDAC4 and neuromuscular junction associated genes) and a
paradoxical increase in the ubiquitin proteasome pathway (31).
These results could suggest that different sets of substrates
are targeted for ubiquitination by MuRF1 under these divergent
atrophy conditions. What mechanisms could accomplish dif-
ferential targeting of substrates under different atrophy condi-
tions? One possibility is that MuRF1 (an E3 ligase) pairs with
different E2 ligases under different atrophy conditions. It has
been shown that the specific E2-E3 pairing can alter the type of
ubiquitin chains that are added to a substrate (42, 60). It is also
possible that different E2-E3 pairings affect the specific sub-
strates that are targeted. Furthermore, it is known that different
proteins can bind to E3 ligases and possibly alter their local-
ization within the cell and substrates (83). One possibility is
that in response to different stressor, MuRF1 has different
binding partners which modify its cellular localization and
 REGULATION OF SKELETAL MUSCLE ATROPHY 279
the cellular localization of the protein, modifying protein-
protein interactions and regulating transcriptional activity (20).
SKELETAL MUSCLE ATROPHY IS A COMPLEX PROCESS

The loss of skeletal muscle mass is the final common

outcome of many diseases and conditions. Over the past twenty
years, a number of proteins and pathways have been identified
as playing a role in the atrophy process (10, 23, 69, 71) (see
Fig. 6). The E3 ubiquitin ligases, MuRF1 and MAFbx, are
particularly interesting because they are relatively muscle spe-
cific, are expressed at low levels under resting conditions, and
are upregulated under all forms of muscle atrophy. It is note-
worthy that MuRF1 and MAFbx are upregulated under diver-
gent atrophy-inducing conditions such as disuse, inflammation,
metabolic stress, oxidative stress, and excess glucocorticoids;
which is not true for all atrophy associated genes. For example,
HDAC4 and Gadd45a are strongly upregulated in response to
denervation and disuse, but not induced in response to gluco-
corticoid treatment (14, 27). There continues to be an ongoing
search to determine whether there is a common set of genes
responsible for muscle atrophy. To answer the question, inves-
tigations should examine multiple divergent atrophy models at
multiple time points during the course of atrophy. It is possible
that different stimuli such as decreased loading, inactivity,
inflammation, metabolic stress or oxidative stress activate an
array of initial factors (e.g., activated GR, FOXOs, ATF4,
NF-␬B, HDAC4, CEBP␤, Smad1/2) that activate some com-
mon pathways (e.g., MuRF1 and MAFbx) and some divergent
pathways (e.g., increased Gadd45a and p21 expression, de-
creased mTORC1 activation, etc.) all leading to the activation
Fig. 5. E3 Ubiquitin ligases expressed in skeletal muscle. There are over 600
E3 ligases in the human genome and they can be broadly classified into two
types: RING finger E3s and the cullin-RING E3 ligase (CRL) superfamily.
MuRF1 belongs to the Ring finger E3 family and MAFbx belongs to the
cullin-RING E3 superfamily. MuRF1 (Trim63) and MAFbx (Fbxo32) repre-
sent just two of the many E3 ligases expressed in skeletal muscle. The majority
of RING finger E3s (A) and cullin-RING E3s (B) expressed in skeletal muscle
have not been studied in any detail.

potential substrates. While the literature suggests that the

primary substrates for MuRF1 are proteins associated with the
thick myofilament (16), this list is most likely incomplete and
may not be accurate since most of the data have been collected
in vitro using E2 ligases that may not associate with MuRF1 in
vivo. Clearly, more investigation is needed to identify the in
vivo substrates of MuRF1 and MAFbx under different atrophy
conditions. Identification of the substrates will provide further
understanding of the mechanisms by which MuRF1 and
MAFbx regulate skeletal muscle size.
MuRF1 and MAFbx represent just two E3 ligases that are
expressed in skeletal muscle (see Fig. 5). To date, only a
handful of E3 ligases have been investigated in skeletal muscle Fig. 6. Multiple pathways lead to muscle atrophy. This diagram illustrates that
under baseline or atrophy-inducing conditions. In addition to skeletal muscle atrophy is initiated by a variety of signals as listed in the blue
MuRF1 and MAFbx, the E3 ligases that have received the most box. These divergent signals activate a variety of transcription factors leading
attention, and been shown to play some role in atrophy, include to the upregulation of a variety of genes and alterations in signaling pathways.
The E3 ligases MuRF1 and MAFbx are highlighted since they are two genes
Fbx30 (MUSA (70),), Trim 32 (17, 46, 47), and Nedd4 (45, that are upregulated in response to divergent signals such as disuse, inflam-
59). A major challenge for the field has been identifying the mation, oxidative stress, and starvation. Other genes such as MUSA1,
substrates targeted for ubiquitination by an individual E3 ligase Gadd45a, and p21 appear to upregulate in response to specific signals.
and then understanding the effect of ubiquitination on the Suppression of the mTORC1 pathways and protein synthesis is a common
response to many atrophy conditions; an exception being denervation. All of
protein. While ubiquitination of a protein can target a protein these pathways lead to a common outcome, the loss of muscle mass, i.e.,
for degradation by the proteasome, it is can also serve other muscle atrophy. A major challenge is to understand how these various
functions such as altering the activity of the protein, altering pathways intersect to induce muscle atrophy.

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280 REGULATION OF SKELETAL MUSCLE ATROPHY
of a common final program to increase the disassembly and
degradation of myofibrils leading to a decrease in muscle fiber
size and muscle atrophy. If this model is correct, then a single
drug would likely not be able to treat all forms of atrophy.
Furthermore, the timing of the treatment will be important. For
example, following disuse and denervation-induced atrophy
MuRF1 increases rapidly and then returns to baseline by 14
days. Therefore, the use of a MuRF1 inhibitor after 14 days
may not be appropriate. However, deletion of MuRF1 contin-
ues to result in muscle sparing after 14 days of disuse or
denervation, suggesting that MuRF1 upregulation initiates spe-
cific signaling cascades that initiate and sustain muscle atro-
phy. Thus, the critical missing data are the identification and
verification of the in vivo substrates for MuRF1 and other E3
ligases which will provide insights into the mechanisms by
which they regulate skeletal muscle mass.
CONCLUSIONS
Skeletal muscle atrophy continues to be a serious conse-
quence of many diseases and conditions for which there is no
treatment. The development of treatments relies on an under-
standing of the molecules and pathways involved in the atro-
phy process. Over the past two decades considerable progress
has been made in our understanding of the molecular and
cellular mechanisms underlying the loss of muscle mass. The
data suggest that while the loss of muscle mass is a common
outcome in many diseases, the pathways leading to the out-
come may vary depending on the initiating signal, i.e., unload-
ing, inactivity, elevated cytokines or malnutrition. Interest-
ingly, the upregulation of the E3 ligases MuRF1 and MAFbx
seems to be a common early event in many divergent atrophy
conditions, e.g., inflammation vs disuse vs starvation. How-
ever, it is clear that upregulation of MuRF1 and MAFbx is not
the complete picture since deletion of these genes does not
result in the complete sparing of muscle mass. The challenge
for the field of muscle biology is to determine the “network” of
pathways involved in muscle atrophy and to understand how
all of the various molecules, transcription factors and signaling
pathways interact to produce muscle atrophy. Identification of
the critical nodes that when suppressed result in a reduction or
blockage of the atrophy process will be necessary for the
successful development of new treatments.
ACKNOWLEDGMENTS
Throughout my career, I have been fortunate to have worked with great
colleagues at the University of California, Los Angeles; University of Cali-
fornia, San Diego; Regeneron Pharmaceuticals; University of California,
Davis; and most recently the University of Iowa. I would like to thank the
many colleagues who have contributed to this research.
GRANTS
This work was supported by grants awarded to S.C.B. by National
Institutes of Health DK075801 AR070031 and the Veterans Administration
1I0RX000673.
DISCLOSURES
Dr. Bodine holds equity in Emmyon, Inc., and serves on the Scientific
Advisory Board.
AUTHOR CONTRIBUTIONS
S.C.B. conceived and designed research, prepared figures, drafted, edited
and revised, and approved final version of manuscript.
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