THE

JOURNAL • RESEARCH • www.fasebj.org

Coordinated regulation of skeletal muscle mass and

metabolic plasticity during recovery from disuse
Anita Kneppers,* Pieter Leermakers,* Nicholas Pansters,* Evelien Backx,† Harry Gosker,* Luc van Loon,†
Annemie Schols,* Ramon Langen,*,1 and Lex Verdijk†,1,2
*Department of Respiratory Medicine and †Department of Human Biology, School of Nutrition and Translational Research in Metabolism
(NUTRIM), Maastricht University Medical Centre+, Maastricht, The Netherlands

ABSTRACT: Skeletal muscle regeneration after disuse is essential for muscle maintenance and involves the regulation
of both mass- and metabolic plasticity–related processes. However, the relation between these processes during
recovery from disuse remains unclear. In this study, we explored the potential interrelationship between the
molecular regulation of muscle mass and oxidative metabolism during recovery from disuse. Molecular profiles
were measured in biopsies from the vastus lateralis of healthy men after 1-leg cast immobilization and after 1 wk
reloading, and in mouse gastrocnemius obtained before and after hindlimb suspension and during reloading (RL-1,
-2, -3, -5, and -8 d). Cluster analysis of the human recovery response revealed correlations between myogenesis and
autophagy markers in 2 clusters, which were distinguished by the presence of markers of early myogenesis,
autophagosome formation, and mitochondrial turnover vs. markers of late myogenesis, autophagy initiation, and
mitochondrial mass. In line with these findings, an early transient increase in B-cell lymphoma-2 interacting protein-
3 and sequestosome-1 protein, and GABA type A receptor-associated protein like-1 protein and mRNA and a late
increase in myomaker and myosin heavy chain-8 mRNA, microtubule-associated protein 1 light chain 3-II:I ratio,
and FUN14 domain-containing-1 mRNA and protein were observed in mice. In summary, the regulatory profiles of
protein, mitochondrial, and myonuclear turnover are correlated and temporally associated, suggesting a coordinated
regulation of muscle mass– and oxidative metabolism–related processes during recovery from disuse.—Kneppers,
A., Leermakers, P., Pansters, N., Backx, E., Gosker, H., van Loon, L., Schols, A., Langen, R., Verdijk, L. Coordinated
regulation of skeletal muscle mass and metabolic plasticity during recovery from disuse. FASEB J. 33, 1288–1298 (2019).
www.fasebj.org
KEY WORDS: remobilization • remodeling • protein turnover • myogenesis • mitophagy

Skeletal muscles are essential to generate the forces for makeup, which are tightly regulated and can rapidly adapt
postural support and physical function. Furthermore, to alterations in mechanical and metabolic demands—
representing half of the body mass and being highly referred to as skeletal muscle plasticity.
metabolically active, skeletal muscle tissue is an important Muscle disuse results from low skeletal muscle me-
site for control of metabolism (1). These functions are chanical loading, such as occurs with bed rest during
supported by the muscles’ biochemical and morphologic hospitalization after surgery or acute illness, with immo-
bilization after a fracture, and with certain chronic dis-
eases. It is well established that disuse leads to a reduction
ABBREVIATIONS: 1-RM, one-repetition maximum; BECN, beclin; BL,
of skeletal muscle strength (2) and mass (2, 3), which re-
baseline; BNIP3, BCL2 interacting protein 3; CSA, cross-sectional area;
FUNDC1, FUN14 domain containing 1; GABARAPL1, GABA type A re- sults from a negative balance in muscle protein turnover
ceptor associated protein like 1; MAP1LC3B, microtubule associated (2, 4). In addition, disuse affects muscle quality, including a
protein 1 light chain 3 b; MSTN, myostatin; MYH, myosin heavy chain; transition from a slow to fast fiber type (5–7), a shift from
MYMK, myomaker; MYOG, myogenin; OPTN, optineurin; PAX, paired
box; PINK1, PTEN-induced putative kinase 1; PRKN, parkin RBR E3
fat oxidation toward glycolysis (8), a decrease in mito-
ubiquitin protein ligase; RL, reloading; SC, satellite cell; SQSTM1, chondrial mass and function (9–12), and a reduction in
sequestosome 1; TBST, Tris-buffered saline with Tween 20; UL, unloading; vascularization (13).
ULK1, unc-51 like autophagy activating kinase 1 The above-mentioned qualitative alterations in re-
1
2
These authors contributed equally to this work. sponse to disuse collectively reduce the capacity for oxi-
Correspondence: Department of Human Biology, Maastricht University, dative metabolism. It has been suggested that these
PO Box 616, 6200 MD Maastricht, The Netherlands. E-mail: lex.verdijk@
maastrichtuniversity.nl metabolic alterations represent an adaptation to a de-
creased energy requirement resulting from a reduction in
doi: 10.1096/fj.201701403RRR
This article includes supplemental data. Please visit http://www.fasebj.org to the level of protein turnover upon disuse (8). On the other
obtain this information. hand, a study showed that the inhibition of disuse-induced

1288 0892-6638/19/0033-1288 © FASEB

mitochondrial alterations prevents the loss of muscle MATERIALS AND METHODS
mass (12). These findings indicate that muscle disuse–
induced mass and metabolic alterations are highly Subjects, study design, and measurements
interdependent.
Although disuse-induced alterations in skeletal muscle Skeletal muscle molecular profiles of 14 healthy young men (age,
mass and function may be part of a normal physiologic 23 6 1 yr; body mass index, 22.9 6 0.6 kg/m2) who participated
in a double-blind, randomized, placebo-controlled intervention
response, the negative implications of muscle disuse at- trial (www.clinicaltrials.gov; NCT01894737) were analyzed. Writ-
rophy for general health, quality of life (14), and even ten informed consent was obtained from all subjects, and the
survival (15–17) have been clearly demonstrated. Both study was approved by the Maastricht University Medical
nutritional interventions and exercise mimetics have been Center+ (Maastricht, The Netherlands) ethics review board (13-
used to prevent muscle disuse atrophy, but none serve as a 3-023) and performed in accordance with the Declaration of
complete substitute for the mechanical and metabolic Helsinki. The study design and primary data were published
stimulation during normal muscle use (18–22). Never- previously (36). In brief, participants were subjected to 7 d of 1-leg
immobilization by means of a full leg cast [unloading (UL)],
theless, in otherwise healthy individuals, a normalization followed by 7 d of remobilization [reloading (RL)]. Subjects were
of muscle mass and function occurs upon reloading after asked to maintain habitual dietary intake and refrain from any
disuse (23, 24). However, the negative consequences of heavy physical exercise during the entire intervention period.
disuse may persist when muscle regeneration is impaired, Their compliance was checked and confirmed by dietary intake
as occurs in elderly (25–27) and chronically ill persons (28). records and activity journals. Furthermore, the subjects con-
To identify and target factors that hamper skeletal muscle sumed standardized meals the evening before all test days.
Needle biopsies from the vastus lateralis of the casted leg were
recovery during resumed physical activity after disuse in
taken after an overnight fast after UL and again after RL. Samples
such persons, a better understanding of the molecular al- were frozen in liquid nitrogen and stored at 280°C until further
terations driving skeletal muscle plasticity during analysis. Furthermore, muscle mass, measured by cross-sectional
reloading-induced recovery is imperative. area (CSA) was assessed by a computed tomographic scan of the
In contrast to the wealth of data on the molecular al- upper legs, muscle strength was assessed with a 1-repetition
terations during skeletal muscle disuse in both humans maximum (1-RM) knee extension test, and body weight was
and animals, insight into the molecular response to measured with a digital scale. None of the subjects performed
progressive resistance-type exercise training or took creatine
reloading to induce skeletal muscle regeneration is less supplements or any medication that would interfere with muscle
abundant and depends mainly on data from studies in metabolism in the 6 mo before the study.
rodents. From these studies, it is clear that protein syn- Only subjects with a complete set of available muscle bi-
thesis signaling related to the control of mRNA translation opsies were included in the current subanalysis (n = 14), of
is increased upon reloading after disuse (27, 29, 30) and whom 5 received placebo and 9 received creatine supple-
that markers of the ubiquitin proteasome system (UPS) mentation. Based on an increase of ,10 mM/kg in muscle total
return to baseline levels during reloading (24, 30). Rather creatine content (36), 4 subjects were considered nonre-
sponders to creatine loading. Furthermore, creatine supple-
than a passive normalization of the protein turnover bal-
mentation did not have any impact on muscle disuse or
ance, several studies suggest a transient activation of subsequent recovery (36).
protein degradation signaling during reloading-induced
recovery, particularly of the autophagy-lysosome path-
way (24, 27, 31, 32). In addition, myogenesis seems to be Animals and study design
activated during skeletal muscle reloading (4, 30, 33).
These reloading-induced molecular alterations are also The animal study was approved by the Institutional Animal Care
implicated in intrinsic remodeling of skeletal muscle. In- Committee of Maastricht University (DEC-2009-074). The study
deed, metabolic alterations take place upon skeletal mus- design has been published (30, 34). In the current subanalysis,
cle reloading, including an activation of processes that only the control (i.e., GSK-3bfl/fl MLC1f-Cre2/2) animals were
regulate mitochondrial biogenesis and function (32, 34, 35) used. In brief, 13-wk-old male C57/Bl6 mice were housed in a
temperature-controlled room (21–22°C) with a 12–12 h light–
and an increase in mitochondrial mass (32, 34). Similar to dark cycle and standard chow pellets and water ad libitum. Mice
the interdependency of mass and metabolic alterations were subjected to either no experimental procedure (baseline, BL;
upon muscle disuse, these processes may be linked during n = 9), 14 d of muscle UL by hindlimb suspension (UL, n = 8), or 1,
reloading-induced recovery. However, such a potential 2, 3, 5, or 8 d of RL after hindlimb suspension (n = 8 per time
relation is largely understudied. In the present study, we point). Mice were euthanized at each time point by injection with
therefore sought to provide insight in the interrelationship pentobarbital sodium, and hindlimb muscles were excised with
standardized dissection methods. Muscles were cleaned of ex-
between the regulation of muscle mass and muscle meta- cess fat and connective tissue, pair weighed on an analytical scale,
bolic plasticity during recovery from disuse. A better un- and snap frozen in liquid nitrogen (biochemical analyses) or
derstanding of these relations is essential for the embedded in Tissue-Tek optimal cutting temperature compound
identification of the molecular origin, and subsequent (Sakura Finetek, Zoeterwolde, The Netherlands) and frozen in
treatment, of impaired remobilization-induced skeletal melting isopentane (histochemical analyses). Samples were
muscle recovery in the elderly and chronically ill. We ex- stored at 280°C until further analysis. The gastrocnemius was
plored these relations by measuring a selected set of indi- used for protein and mRNA analyses, as its response to hindlimb
UL is pronounced and well characterized in studies (37–39).
cators and molecular regulators and mediators of muscle Furthermore, it has a fiber type composition and distribution that
mass– and oxidative metabolism–related processes and is representative of many muscles in the hindlimb and is similar
studied their interrelationship during reloading-induced to the fiber type composition observed in the human vastus
recovery after disuse in both humans and rodents. lateralis.

COORDINATED MUSCLE MASS AND PLASTICITY REGULATION 1289

Protein isolation and Western blot analysis Transcriptor cDNA synthesis kit (Roche, Basel Switzerland),
according to the manufacturer’s instructions. Primers were
For protein analysis, frozen mouse gastrocnemius was powdered designed based on Ensemble transcript sequences and ordered
and lysed in a whole-cell lysis buffer (50 mM Tris, 150 mM NaCl, from MilliporeSigma, with primer details shown in Supple-
10% glycerol, 0.5% Nonidet P40, 1 mM EDTA, 500 mM Na3VO4, mental Table S2. qPCR reactions contained Sensimix Sybr Rox
500 mM NaF, 100 mM b-glycerophosphate, 100 mM Na4P2O7, (GC Biotech, Alphen aan den Rijn, The Netherlands) and primer
1 mM DTT, 10 mg/ml leupeptin, and 1% aprotinin) for 1 h on a mix and were run in a 384-well white opaque plate (Roche Di-
tube rotator at 4°C, followed by 30 min of centrifugation at 14,000 agnostics, Indianapolis, IN, USA) on a LightCycler 480 system
g at 4°C. Lysates were aliquoted in sample buffer [0.25 M Tris- (Roche Diagnostics). Melting curves were analyzed to verify
HCl, 8% (w/v) SDS, 40% (v/v) glycerol, 0.4 M DTT, and 0.02% specificity of the amplification, and relative quantity of the tar-
(w/v) bromophenol blue], boiled for 5 min at 95°C, and stored at gets was assessed by LinRegPCR software (v.2014.8; http://www.
280°C. Human vastus lateralis biopsies were homogenized in hartfaalcentrum.nl) (Heart Failure Research Centre, Amsterdam,
lysis buffer (20 mM Tris-HCl, 1% Nonidet P40, 5 mM EDTA, The Netherlands). Study-specific reference genes (Supplemental
2 mM Na3VO4, 100 mM NaF, 10 mM Na4P2O7, 10 mg/ml leu- Table S2) were used to calculate a GeNorm correction factor (https://
peptin, 10 mg/ml aprotinin, 3 mM benzamidine, and 1 mM genorm.cmgg.be/), which was used to normalize expression levels of
PMSF) using an Ultra-Turrax (IKA Works, Wilmington, NC, the target genes.
USA). Lysates were centrifuged for 10 min at 10,000 g at 4°C.
Lysates were aliquoted in sample buffer [0.35 M Tris-HCl, 10% Immunohistochemistry
(w/v) SDS, 30% (v/v) glycerol, 0.6 M DTT, and 0.02% bromo-
phenol blue], boiled for 5 min at 100°C, and stored at 280°C.
OCT-embedded, frozen mouse gastrocnemius was cut (7 mm),
Total protein concentration was determined with the Pierce BCA
defrosted, and air dried at room temperature. Tissue was fixed
Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA,
with 4% paraformaldehyde and permeabilized with 0.2% Triton
USA), according to the manufacturer’s instructions.
X-100 in PBS. Subsequently, samples were treated for 60 min with
Ten micrograms protein was separated on a Criterion XT 3% H2O2, blocked for 60 min in blocking solution [5% (v/v) goat
Precast Bis-Tris Gel (Bio-Rad, Hercules, CA, USA) in XT-3-(N- serum, 2% (w/v) bovine serum albumin, 1:40 mouse-on-mouse
morpholino)propanesulfonic acid or XT-2-[N-morpholino]etha- blocking reagent (MOM; Vector Laboratories)] at room temper-
nesulfonic acid running buffer (Bio-Rad) by gel electrophoresis. ature, and incubated overnight at 4°C with primary antibodies
Proteins were transferred to a 0.45 mm nitrocellulose membrane directed against laminin and paired box (PAX)-7 (1:250) (Sup-
(Bio-Rad) by electroblotting at 100 V for 60 min in transfer buffer plemental Table S1). Samples were incubated in horseradish
[25 mM Tris, 192 mM glycine, 20% (v/v) methanol]. For total peroxidase–conjugated streptavidin and goat anti-rabbit 555 (1:
protein detection, membranes were stained with PonceauS so- 200) for 60 min at room temperature and subsequently for 7 min
lution (0.2% PonceauS in 1% acetic acid; MilliporeSigma, Bur- in tyramide working solution and reaction stop reagent,
lington, MA, USA) for 3–5 min, washed in double-distilled H2O according to the manufacturer’s protocol (Tyramide SuperBoost
for 30 s on a shaker, and imaged with the Amersham imager Kit; Thermo Fisher Scientific). Nuclei were stained with DAPI,
600RGB (GE Healthcare, Chicago, IL, USA). The membranes after which cover slips were mounted with Mowiol (Milli-
were blocked for 1 h at room temperature in Tris-buffered saline poreSigma). After staining, images were digitally captured with
with Tween 20 [TBST; 20 mM Tris, 137 mM NaCl, 0.1% (v/v) fluorescence microscopy (Olympus IX-81; Olympus, Hamburg,
Tween 20 (pH 7.6)] containing 5% (w/v) nonfat dry milk Germany) connected to a digital camera (EXi Aqua; QImaging,
(Campina, Eindhoven, The Netherlands). The membranes were Surrey, BC, Canada) at 3400 magnification. Image processing
washed in TBST, followed by overnight incubation at 4°C with and quantitative analyses were performed with ImageJ (National
primary antibody diluted in TBST with 3% bovine serum albu- Institutes of Health, Bethesda, MD, USA).
min (Supplemental Table S1). After a wash in TBST, membranes
Laminin staining was used to determine the cell borders.
were incubated with a peroxidase-conjugated secondary anti-
Within each image, the mean fiber CSA, the number of myonuclei
body solution (Vector Laboratories, Burlingame, CA, USA) for 1
per fiber, and the mean fiber CSA per nucleus were quantified.
h at room temperature, and targets were visualized by chem-
Satellite cells (SCs) were manually counted and were defined as
iluminescence (Supersignal West PICO or FEMTO Chemilumi-
PAX7+ nuclei located within the basal lamina.
nescent substrate; Thermo Fisher Scientific), according to the
manufacturer’s instructions, and detected with the Amersham
Imager 600RGB. Signals were quantified with Image Quant TL Data analysis and statistics
software (GE Healthcare). Samples from each group were dis-
tributed within and between blots, to allow for correction of To address the relation between alterations in skeletal muscle
between-session variation (40), and protein expression and mass– and oxidative metabolism–related processes during RL
phosphorylation levels were corrected for protein loading after disuse, clustering of human skeletal muscle biochemical and
assessed by PonceauS. physiologic parameters was performed with the SciPy (v.0.19.1)
toolkit (41). The RL response (RL–UL) per parameter was
RNA isolation and RT-qPCR expressed as the percentage change from UL. The Spearman
correlation coefficient between each pair of relative RL responses
For RNA analysis, powdered mouse gastrocnemius was lysed in (i.e., Rj,k) was computed and subsequently transformed to Eu-
RLT solution containing 1% 2-ME. Total RNA was extracted clidean distance (Dj,k) with (Eq. 1):
using an on-column RNA isolation kit (Qiagen, Hilden, Ger- qffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffiffi
 
many) according to the manufacturer’s instructions and stored at Dj;k ¼ 1 2 Rj;k  (1)
280°C. Human vastus lateralis biopsies were lysed in Trizol
(Thermo Fisher Scientific). Total RNA was isolated by phenol- The resulting Euclidean distance matrix was hierarchically clus-
chloroform extraction and subsequent precipitation from the tered based on the between-cluster average linkage using SciPy’s
aqueous phase with glycogen-containing isopropanol. RNA was cluster.hierarchy.linkage module and visualized using the clus-
reconstituted in nuclease-free H2O and stored at 280°C. termap module within Seaborn v.0.8.1 (42). Differences in the
RNA concentration was determined with a spectrophotom- distribution of markers between clusters were tested by Pear-
eter (Nanodrop ND-1000 UV-Vis; Thermo Fisher Scientific). son’s x2 test with post hoc analysis based on adjusted residuals
RNA (400 ng/sample) was reverse transcribed into cDNA with a with Bonferroni correction.

1290 Vol. 33 January 2019 The FASEB Journal x www.fasebj.org KNEPPERS ET AL.
 The RL response per parameter of paired data (human bi- myogenesis (i.e., proliferation). Moreover, most of the
opsies) was tested by paired-samples t test. The time-dependent autophagy markers in cluster 3 [i.e., optineurin (OPTN)
RL response (mouse muscle) was tested by 1-way ANOVA with mRNA, and microtubule associated protein 1 light chain 3
Dunnett’s post hoc test, using UL as a reference group. Data are
b (MAP1LC3B)-II, MAP1LC3B-I, and beclin (BECN)-1
expressed as means 6 SEM, unless indicated otherwise. Statistical
analyses were performed with SPSS Statistics (v.22.0; IBM, protein] are indicators of autophagosome formation,
Armonk, NY, USA). whereas most of the autophagy markers in cluster 4 [i.e.,
phosphorylated unc-51–like autophagy-activating kinase
1 (ULK1) ser556, pULK1ser556:ULK1 ratio, pULK1ser758,
RESULTS pULK1ser758:ULK1 ratio, GABA type A receptor–
associated protein-like 1 (GABARAPL1), and sequestosome
One week of remobilization after leg immobilization–
1 (SQSTM1) protein] are indicators of autophagy initiation.
induced atrophy tended to increase quadriceps CSA
(Table 1). However, neither quadriceps CSA nor muscle
force measured by 1-RM had returned to preimmobilization Induction of late myogenic markers after
baseline levels, indicating that recovery was still ongoing. 5 d of RL in mice

Regulators and mediators of myogenesis, The distinct clustering of early (cluster 4) and late (cluster
autophagy, and mitochondrial mass and 3) myogenesis markers upon remobilization in human
dynamics correlate during recovery from subjects may reflect a time-dependent expression pattern
muscle disuse in humans of these markers, as previously observed for early myo-
genesis markers by Pansters et al. (30). This RL time-course
To address whether there is a correlation between alterations mouse study was therefore used to address those time-
in skeletal muscle mass– and oxidative metabolism– dependent expression patterns. In this study, UL de-
related processes during RL after disuse, we measured creased the myofiber CSA, myonuclear domain, and
the protein and mRNA expression of several regulators number of nuclei per fiber (Fig. 2A–C). Ongoing recovery
and mediators of protein turnover, mitochondrial turn- was indicated by an incomplete normalization of CSA and
over, and myonuclear turnover, before and after 7 d of myonuclear domain up to RL-8, and the number of nuclei
RL in humans and clustered the correlations between per fiber was significantly restored at RL-3 and -8. This
relative RL responses based on similarity. From this, 4 restoration of the myonuclei was paralleled by a tendency
clusters of parameters were identified (Fig. 1). toward an increased number of SCs at RL-3 (Fig. 2D, E).
Cluster 1 appeared enriched in markers of protein To assess whether late myogenesis markers indeed
turnover signaling, whereas cluster 2 seemed to be display a distinct temporal expression pattern, we mea-
enriched in markers of muscle mass and function (Sup- sured markers of SC fusion and early myofiber re-
plemental Table S3). Furthermore, two major clusters (3, 4) generation in mouse gastrocnemius at 1, 2, 3, 5, and 8 d of
occurred in which markers of myogenesis and autophagy RL after hindlimb suspension. At RL-1 and -2, Myh9 was
were strongly represented (Supplemental Table S3). This transiently increased and gradually normalized at RL-5
strong representation of myogenesis and autophagy and -8 (Fig. 3). The expression of another, novel fusion
markers was accompanied by a high percentage of marker, myomaker (Mymk), was strongly but transiently
markers of mitochondrial turnover and mitochondrial induced at RL-5. Furthermore, Myh8 was induced at RL-5
dynamics in cluster 3, whereas cluster 4 was concomi- and -8. Expression of the muscle-specific gene creatine
tantly enriched in markers of mitochondrial mass and kinase (Ckm) was reduced upon UL, and was transiently
appeared enriched in markers of mitochondrial dynamics, further reduced at RL-1, -2, and -3, whereas expression of
and to some extent, protein turnover (Supplemental Table the negative regulator of muscle mass Mstn was reduced at
S3). Furthermore, most of the myogenesis markers repre- RL-5 and -8.
sented in cluster 3 [i.e., myogenic factor (MYF)-5, myosin
heavy chain (MYH)-8, myogenin (MYOG), and MYH9 Parallel modulation of autophagy and
mRNA] are associated with late stages of myogenesis (i.e., myogenesis in mice
differentiation and fusion), whereas most of the myo-
genesis regulators and markers represented in cluster 4 To address whether the temporal expression patterns of
[i.e., myostatin (MSTN) protein, and cyclin D1, PAX7, and early and late myogenesis markers coincide with a tem-
cadherin 15 mRNA] are associated with early stages of poral regulation of autophagy (as suggested from clusters

TABLE 1. Response to immobilization

Parameter Preimmobilization Postimmobilization Remobilization

Body weight (kg) 70.9 6 3.3 72.0 6 3.2 71.7 6 3.1

Quadriceps CSA (mm2) 8004 6 320 7562 6 303*** 7671 6 315**,#
1-RM (kg) 57 6 3 50 6 2** 52 6 2*

Data are expressed as means 6 SEM. *P , 0.05, **P , 0.01, ***P , 0.001 vs. preimmobilization,
#
P , 0.1 vs. postimmobilization.

COORDINATED MUSCLE MASS AND PLASTICITY REGULATION 1291

Figure 1. A correlation heatmap and cluster dendrogram of biochemical and functional parameters during skeletal muscle
recovery from disuse in humans. Each square represents the Spearman correlation coefficient between the relative RL responses
of parameters displayed on the x- and y-axes, with colors indicating the strength and direction of the correlation.

3 and 4), mRNA and protein levels of indicators of auto- but transient increase at RL-1 compared to UL, although
phagosome formation and markers of autophagy capacity Gabarapl1 mRNA was initially reduced upon UL (Figs. 4
and autophagy initiation were measured. and 5). The mRNA expression of Optn, Becn1, and auto-
The relative protein expression of Map1lc3b-I displayed phagy related-7 were unaltered during RL (Fig. 5).
an acute but transient increase at RL-1, -2, and -3, whereas To further address a main regulatory step in the con-
Map1lc3b-II levels remained largely unaltered (Fig. 4). The version of changes in autophagy capacity into flux, the
Map1lc3b-II:I ratio was decreased at RL-1, but sub- activating and inhibiting phosphorylation of Ulk1 was
sequently normalized, and even increased at RL-8. Fur- assessed. Both absolute and relative activating Ulk1
thermore, the relative mRNA expression of Map1lc3b was phosphorylation (ser555) were increased during RL, up to
decreased at RL-2, -3, and -5, and seemed to be partially RL-3 (Fig. 6). However, in parallel, both absolute and rel-
normalized at RL-8 (Fig. 5). In contrast, Gabarapl1 and ative inhibiting Ulk1 phosphorylation (ser757) were in-
Sqstm1 protein and mRNA expression displayed an acute creased during RL, up to RL-5.

1292 Vol. 33 January 2019 The FASEB Journal x www.fasebj.org KNEPPERS ET AL.
 Figure 2. Changes in myofiber
CSA, myonuclear domain (MD),
and the number of myonuclei
and SCs during muscle disuse and
recovery in mice. A–D) Gastroc-
nemius muscles were collected at
baseline (BL), after 14 d of UL, or
after 1, 3, or 8 d of RL, for
immunohistochemical determi-
nation of mean muscle fiber
CSA (A); MD (B); mean number
of myonuclei (C); and mean
number of PAX7+ SCs per mus-
cle fiber (D). E) Representation
of immunohistochemical detec-
tion of laminin (red) and PAX7
(green), and nuclei stained with
DAPI (blue). Arrowheads: PAX7+
SCs, arrows: myofiber nuclei. #P
, 0.1, *P , 0.05, **P , 0.01, vs.
BL, and $P , 0.05 vs. UL.

Temporal induction of mitophagy during increased at RL-1, subsequently returned to UL values,

recovery from disuse in mice and decreased at RL-5 and -8 (Fig. 7). Furthermore, FUN14
domain containing 1 (Fundc1) protein expression was in-
Next to the predominance of markers of autophagosome creased at RL-8 compared to UL, although an initial de-
formation in cluster 3, this cluster also appeared to be crease was observed upon UL. Corresponding to its
enriched in markers of mitophagy. We therefore assessed change in protein abundance, Fundc1 mRNA expression
the temporal expression of mitophagy markers during was increased at RL-8 (Fig. 8). Furthermore, Bnip3l mRNA
recovery from disuse in mice. expression was decreased, and phosphatase and tensin
No significant alterations in protein expression of BCL2 homolog (PTEN)-induced putative kinase (Pink)-1 tended
interacting protein 3 like (Bnip3l) or parkin RBR E3 ubiq- to be decreased upon remobilization, up to d RL-8, but no
uitin protein ligase (Prkn) were observed during recovery changes in the expression of Prkn mRNA were observed.
after disuse. However, the protein expression of Bnip3
DISCUSSION

Alterations in the regulation of both muscle mass and

metabolic plasticity during recovery from disuse have
been reported, but the interaction between these processes
is understudied. Hence, we explored this potential in-
teraction by measuring established molecular regulators
and mediators of processes that determine muscle mass
and metabolism in human and mouse muscle and showed
that molecular markers of myogenesis, autophagy, and
mitochondrial mass and dynamics change coordinately
during recovery from disuse.
In the current human study, 1 wk of remobilization after
leg immobilization-induced atrophy increased quadriceps
CSA compared to postimmobilization, but did not return
Figure 3. Temporal changes in mRNA expression of markers quadriceps CSA or muscle force measured by 1-RM to
and mediators of late myogenesis upon recovery from muscle
disuse in mice. mRNA expression of MYH9, MYMK, MYH8, preimmobilzation baseline levels. This finding is in line
creatine kinase, and MSTN was assessed in gastrocnemius with those in studies that showed an incomplete nor-
upon recovery from 14 d hindlimb suspension. #P , 0.1, *P , malization of CSA (43, 44) and muscle strength (44) upon
0.05, **P , 0.01, ***P , 0.001 vs. UL. 1 wk of recovery after immobilization and a complete

COORDINATED MUSCLE MASS AND PLASTICITY REGULATION 1293

 time-course study in mice was thus instrumental in the in-
depth exploration of the time-dependent molecular regu-
lation of muscle mass and metabolic plasticity during
disuse-induced atrophy recovery.
Similar to the study in humans, mice displayed an in-
complete normalization of muscle weight (30) and muscle
fiber CSA, which supports ongoing recovery over 8 d of
RL. Three days of RL after hindlimb suspension tran-
siently increased the number of PAX7+ SCs per fiber. This
finding is in line with previous studies assessing SC
numbers after relative short-term remobilization in ro-
dents (50–52) and humans (46) and may explain the un-
altered number of SCs upon longer term remobilization
(53). In accordance with the early induction of SC pro-
liferation, our lab showed in another study an early and
potentially biphasic induction of markers and mediators of
Figure 4. Temporal changes in protein expression of auto- the initial steps in myogenesis in this model (30). In the
phagosome formation indicators upon recovery from mus- current work, we showed for the first time that, sub-
cle disuse in mice. Protein expression of MAP1LC3B-I, sequent to this increase in early myogenesis markers, the
MAP1LC3B-II, MAP1LC3B-II:I, GABARAPL1, and SQSTM1 markers and mediators of late stages of myogenesis Mymk
was assessed in gastrocnemius upon recovery from 14 and Myh8 are increased toward the end of the 8 d RL
d hindlimb suspension. #P , 0.1, *P , 0.05, **P , 0.01,
***P , 0.001 vs. UL. phase. Furthermore, in line with refs. 54 and 56, there was a
partial restoration of the number of nuclei per fiber after
3 d of RL, which supports the notion that SCs functionally
normalization of muscle mass and strength after 2 wk of contribute to skeletal muscle recovery after disuse (57).
recovery (25, 44–47). This result shows that after 1 wk of Indeed, because the myonuclear domain was unaltered
remobilization, skeletal muscle recovery is still ongoing, during remobilization, the increase in myonuclear content
and thus allows us to study the processes that are asso- is expected to result in an increase in muscle fiber CSA.
ciated with recovery. However, as this change in fiber CSA is not yet signifi-
We detected substantial interindividual variation cantly detectable, we conclude that muscle fiber CSA is
in the human molecular responses to RL (Supplemental incompletely restored up to 8 d of remobilization. These
Fig. S1), which was not affected by correction for crea- findings could be speculated to result from a lag time be-
tine supplementation. We exploited this variation to as- tween myonuclear accretion and a functional contribution
sess the relation between molecular RL responses. Based of the newly incorporated nucleus to protein synthesis or
on hierarchical cluster analysis of the correlations be- to reflect a role for myonuclear accretion in muscle fiber
tween relative RL responses, we identified 4 clusters of metabolic plasticity. Despite the incomplete normalization
parameters. The coappearance of markers and mediators of muscle fiber CSA and myonuclear domain over the time
of classic muscle mass– and oxidative metabolism–related course examined in this mouse study, their recovery
processes within clusters 3 and 4 may point at an in- seemed to start as early as RL-1. This is in agreement with
terdependency or joint regulation of these processes dur-
ing recovery from atrophy. Although we cannot exclude
creatine supplementation as a potential confounding fac-
tor and a modulatory effect of creatine supplementation
on certain molecular processes has been suggested (48, 49),
there is no indication of an effect of creatine supplemen-
tation on the interrelationship between the regulatory
processes associated with recovery from muscle atrophy,
and correction for creatine supplementation does not alter
the pattern of correlations between RL effects. Markers
that regulate and mediate different stages of myogenesis
and autophagy displayed a distinct clustering, which may
be related to differences in their temporal expression
patterns. Between-subject asynchronicity in such tempo-
ral expression patterns may contribute to the observed
variation, which—despite its applicability in the assess-
ment of between-variable associations—limits the statis-
tical power to detect relevant changes on a group level. Figure 5. Temporal changes in mRNA expression of auto-
phagy-related genes upon recovery from muscle disuse in
Furthermore, interindividual asynchronicity, combined mice. mRNA expression of MAP1LC3B, GABARAPL1, SQSTM1,
with a single time-point observation during recovery in OPTN, BECN1, and autophagy related (ATG)-7 was assessed in
humans, complicates the synthesis of a univocal conclu- gastrocnemius upon recovery from 14 d hindlimb suspension.
sion on the molecular RL response. The high-resolution **P , 0.01, ***P , 0.001 vs. UL.

1294 Vol. 33 January 2019 The FASEB Journal x www.fasebj.org KNEPPERS ET AL.
 Despite our comprehensive analysis of autophagy
regulation, we did not measure autophagy flux and
therefore cannot draw an unambiguous conclusion re-
garding the net effect of RL on the autophagy flux itself.
Furthermore, alterations in expression levels of several
autophagy regulators and mediators seem contradictory,
which emphasizes the complexity of autophagy regula-
tion during recovery after disuse. We speculate that this
complexity reflects the degradation of specific targets (e.g.,
mitochondria), which is in accordance with a possible in-
volvement of autophagy in skeletal muscle metabolic
plasticity in addition to its classic role in muscle mass
regulation (67, 68). Recent papers suggest a crucial role for
autophagy, and more specifically, mitophagy, in skeletal
muscle functional regeneration (69, 70).
During recovery after disuse in mice, the gene expres-
sion of the mitochondrial damage-related mitophagy
Figure 6. Temporal changes in protein expression of markers of regulator Pink1 tends to be decreased, whereas Prkn is
autophagy initiation upon recovery from muscle disuse in mice. unaltered. In addition, Prkn protein expression is un-
Protein expression of ULK1, p-ULK1(ser555), p-ULK1(ser555): altered during remobilization, although it should be noted
ULK1 ratio, p-ULK1(ser757), and p-ULK1(ser757):ULK1 ratio was
that this mitophagy pathway is also modulated by post-
assessed in gastrocnemius upon recovery from 14 d hindlimb
suspension. *P , 0.05, **P , 0.01, ***P , 0.001 vs. UL. translational modifications (71), which were not assessed
in this study. Receptor-mediated mitophagy via BNIP3L,
BNIP3, and FUNDC1 can be induced by both hypoxia and
the previously observed acute and sustained increase in increased oxidative stress (72), of which an increase during
protein synthesis signaling (30, 58–60) and increased pro- RL has been documented (73, 74). Bnip3 protein expression
tein synthesis rates upon RL (61). This anabolic state pre- transiently increased, and subsequently decreased during
cedes myonuclear accretion, which supports the notion recovery, which parallels the previously reported alter-
that hypertrophy can occur to some extent independent of ations in Bnip3 mRNA expression in this mouse study (30),
SCs (62). Furthermore, previous studies have consistently and the increase in Bnip3 protein expression reported by
shown a suppression of mediators of the UPS during re- Kang and Ji (74). In contrast, Bnip3l protein expression is
covery from disuse (27, 30, 44, 52, 59, 60, 63–65), whereas a unaltered upon recovery after disuse, whereas Bnip3l
transient increase in proteasome activity (27, 61) and mRNA expression displays a persistent decrease up to RL-
ubiquitin-conjugated proteins (60) have been observed 5. The mRNA and protein expression of Fundc1 is specif-
simultaneously. ically induced at RL-8, suggesting a late-phase response.
In this study, we conducted a comprehensive analysis These molecular alterations point toward a temporal
of markers of autophagosome formation, autophagy ca- induction of mitophagy both acutely and after 8 d of re-
pacity, and autophagy initiation. From this, we conclude covery after disuse. Nevertheless, previous data suggest
that autophagy is actively regulated both acutely, as well a normalization of mitochondrial content upon several
as after 5 and 8 d of RL, which appears to parallel the days of RL (34, 73–75), likely driven by an increase in
temporal regulation of myonuclear accretion. We showed
an acute but transient increase in the autophagosome
formation markers Map1lc3b-I, Gabarapl1, and Sqstm1
protein, and a decrease and subsequent increase in the
Map1lc3b-II:I ratio that parallels the regulation of late
stages of myogenesis. In line with our data, previous
studies showed an increase in Sqstm1 protein expression
(27) and an increase in Map1lc3b-II:I ratio (31). In addition,
White et al. (63) reported an unaltered Map1lc3b-II:I ratio
after 14 d of RL, potentially reflecting a normalization of
autophagy signaling upon completed recovery. Further-
more, in line with previous studies (59, 66), we showed a
transient decrease in the mRNA expression of the auto-
phagy capacity indicator Map1lc3b and a transient increase
in the mRNA expression of Gabarapl1 relative to UL. In
addition to the reported increase in Ulk1 inhibiting phos-
phorylation (27, 29), we report a concomitant induction Figure 7. Temporal changes in protein expression of
mitophagy regulators and mediators upon recovery from
of Ulk1 inhibiting and activating phosphorylation. This muscle disuse in mice. Protein expression of BNIP3L, BNIP3,
temporal regulation of autophagy initiation resembles the FUNDC1, and PRKN was assessed in gastrocnemius upon
expression pattern of early myogenesis markers observed recovery from 14 d hindlimb suspension. #P , 0.1, *P , 0.05,
by Pansters et al. (30). ***P , 0.001 vs. UL.

COORDINATED MUSCLE MASS AND PLASTICITY REGULATION 1295

 technical assistance in performing the molecular analyses, and
Roy Haast (Maastricht Centre for Systems Biology, Maastricht
University) for support in conducting the cluster analysis. The
original work from which human muscle biopsies were
obtained (36) was funded by TI Food and Nutrition, a
public–private partnership for precompetitive research in food
and nutrition; and the mouse study was funded by Grant NAF
3.2.07.017 from the Lung Foundation/Netherlands Asthma
Foundation and the transnational University Limburg (tUL).
For the current work, the researchers had sole responsibility
for the study design, data collection and analysis, decision to
publish, and preparation of the manuscript. The authors
declare no conflicts of interest.

AUTHOR CONTRIBUTIONS
Figure 8. Temporal changes in mRNA expression of mitoph-
agy-related genes upon recovery from muscle disuse in mice. A. Kneppers, P. Leermakers, H. Gosker, L. van
mRNA expression of BNIP3L, BNIP3, FUNDC1, PINK1, and Loon, A. Schols, R. Langen, and L. Verdijk designed the
PRKN was assessed in gastrocnemius upon recovery from 14 d research; A. Kneppers, P. Leermakers, E. Backx, and N.
hindlimb suspension. #P , 0.1, *P , 0.05, **P , 0.01, ***P , Pansters performed the research; A. Kneppers analyzed
0.001 vs. UL. the data; A. Kneppers, P. Leermakers, R. Langen, and L.
Verdijk wrote the manuscript; and all authors read and
mitochondrial biogenesis (34, 73). The induction of approved the final manuscript.
mitophagy in the presence of a recovery of mitochondrial
content suggests the induction of mitochondrial remod-
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Physiol. Endocrinol. Metab. 303, E1335–E1347 Accepted for publication July 30, 2018.

1298 Vol. 33 January 2019 The FASEB Journal x www.fasebj.org KNEPPERS ET AL.