Articles in PresS. J Appl Physiol (July 21, 2016). doi:10.1152/japplphysiol.00687.2015 1 1
Morphological and molecular aspects of immobilization-induced muscle atrophy
2
in rats at different stages of postnatal development: the role of autophagy
3 4
Camila Silva Foresto1,4, Sílvia Paula-Gomes3,4, Wilian Assis Silveira2, Flávia
5
Aparecida Graça2, Isis do Carmo Kettelhut2,3, Dawit Albieiro Pinheiro Gonçalves2,3,5,
6
Ana Claudia Mattiello-Sverzut1,5
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Departments of 1Biomechanics, Medicine and Rehabilitation of the Locomotor
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Apparatus,
9
School, University of São Paulo, Brazil. These authors contributed equally to this
10
2
Physiology and
3
Biochemistry/Immunology, Ribeirão Preto Medical
work (4first and 5senior authors).
11 12 13
Correspondence to: Dawit Albieiro Pinheiro Gonçalves
14
Departments of Physiology and Biochemistry & Immunology,
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Ribeirão Preto Medical School, USP
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14049-900 Ribeirão Preto, SP, BRAZIL
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FAX: 0055 (16) 33150219
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Tel.: 0055 (16) 33153213
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E-mail address:
[email protected]
20 21 22
Abbreviated title: Autophagy’s role in immobilized muscle
23 24 25
Copyright © 2016 by the American Physiological Society.
2 26
ABSTRACT
27
Muscle loss occurs following injury and immobilization in adulthood and childhood,
28
which impairs the rehabilitation process, however, far fewer studies have been
29
conducted analyzing atrophic response in infants. This work investigated first the
30
morphological and molecular mechanisms involved in immobilization-induced
31
atrophy in soleus muscles from rats at different stages of postnatal development [i.e.,
32
weanling (WR) and adult (AR) rats] and, second, the role of autophagy in regulating
33
muscle plasticity during immobilization. Hindlimb immobilization for 10 days
34
reduced muscle mass and fiber cross-sectional area, with more pronounced atrophy in
35
WR, and induced slow-to-fast fiber switching. These effects were accompanied by a
36
decrease in markers of protein synthesis and an increase in autophagy. The Ub-ligase
37
MuRF1 and the ubiquitinated proteins were upregulated by immobilization in AR
38
while the autolysed form of μ-calpain was increased in WR. To further explore the
39
role of autophagy in muscle abnormalities, AR were concomitantly immobilized and
40
treated with colchicine, which blocks autophagosome-lysosome fusion. Colchicine-
41
treated immobilized muscles had exacerbated atrophy and presented degenerative
42
features. Despite Igf1/Akt signaling was downregulated in immobilized muscles from
43
both age groups, Foxo1 and 4 phosphorylation was increased in WR. In the same
44
group of animals, Foxo1 acetylation and Foxo1 and 4 content was increased and
45
decreased, respectively. Our data show that muscle disorders induced by 10-day-
46
immobilization occurs in both age-dependent and -independent manners, an
47
understanding that may optimize treatment outcomes in infants. We also provide
48
further evidences that the strong inhibition of autophagy may be ineffective for
49
treating muscle atrophy.
3 50
Keywords muscle atrophy, fiber type, Igf1/Akt/Foxo signaling, protein metabolism,
51
autophagy.
52 53
NEW & NOTEWORTHY STATEMENT
54
Immobilization induces muscle maladaptations at different stages of postnatal
55
development, but the cellular mechanisms involved in such effects are unclear. Our
56
data show that the alterations in muscle proteostasis during immobilization occur in
57
age-independent and -dependent manners and muscle disorders are aggravated by
58
autophagy blockade with colchicine, inducing a myopathic profile. This
59
understanding may help us optimize treatment outcomes in immobilized adults and
60
infants and encourages the test of different autophagy inhibitors in muscle atrophy.
61 62
INTRODUCTION
63
Among several clinical settings, traumatic orthopedic injury is one of the leading
64
causes of morbidity and mortality of both adults (32) and children (16). Skeletal
65
muscle atrophy occurs frequently following injury and immobilization, which results
66
in significant loss of force production and has a profound impact on rehabilitation
67
process (2, 31, 42). Unfortunately, the treatment of muscle wasting under these
68
conditions remains an unsolved problem, especially during childhood, in part, due to a
69
lack of understanding of the cellular and molecular mechanisms responsible for the
70
induction and maintenance of muscle loss.
71
Muscle atrophy can affect specific fiber types and is frequently accompanied by a
72
fiber type switching. For instance, muscle disuse, such as sciatic denervation or hind
73
limb unloading, causes a more pronounced atrophy in slow, oxidative muscles
74
enriched in type I fiber (e.g., soleus) with a slow-to-fast fiber type switching (36, 37).
4 75
The mechanisms by which disuse induces muscle plasticity are uncertain and may
76
involve a decrease in the rate of protein synthesis (55) and/or an increase in protein
77
breakdown (7, 54). Skeletal muscle contains at least three systems for degrading
78
proteins: Ca2+-dependent, ubiquitin (Ub)–proteasome (UPS), and autophagy-
79
lysosome (ALS) proteolytic systems. Ca2+-dependent system includes two ubiquitous
80
well-characterized cysteine proteases, μ-calpain and m-calpain, and their specific
81
endogenous inhibitor, calpastatin (18). The contribution of this proteolytic system to
82
muscle plasticity was evaluated in transgenic mice with muscle-specific
83
overexpression of calpastatin when submitted to hind limb unloading. These
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transgenic mice were partially resistant to atrophy and completely prevented the
85
switch from a slow- to fast-twitch fiber type, which normally occurs in muscle
86
unloading (53).
87
Similar to the inhibition of Ca2+-dependent proteolysis, the in vivo blockade of
88
proteasome by the treatment with VelcadeTM has partially prevented muscle atrophy
89
by denervation and cast immobilization of the hind limb (1, 23). UPS usually
90
degrades the majority of intracellular proteins and consists of ordered actions of
91
enzymes that tag proteins with Ub for degradation (24). Among several Ub-ligases,
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Atrogin-1/MAFbx and MuRF1 are muscle-specific enzymes required to link chains of
93
Ub onto proteins during muscle atrophy (5), which leads to the recognition and
94
degradation of ubiquitinated proteins by proteasome. Unlike the UPS, ALS is
95
primarily responsible for the degradation of most long-lived or aggregated proteins
96
and cellular organelles. The main characteristic of ALS is the formation of
97
autophagosome, which envelops substrates and delivers them to the lysosome for
98
breakdown (46). Among the autophagy proteins, LC3 is critical for the elongation of
99
isolation membranes and is localized to preautophagosomes and autophagosomes,
5 100
making this protein a specific readout for autophagy (22). Despite the expression of
101
several components and the activity of ALS have been found to be upregulated in
102
muscle by a variety of atrophy situations (46, 59), muscle-specific deletion of the
103
crucial autophagy gene Atg7 resulted in profound muscle wasting in basal condition
104
and during denervation and fasting (29). Thus, the protective role of ALS inhibition in
105
muscle atrophy conditions remains unclear.
106
Interestingly, the activities of both UPS and ALS are upregulated by a common
107
family of transcription factors, termed Forkhead box class O (Foxo) (28, 45). At basal
108
state, the phosphorylation of Foxo by Akt, a central kinase that lies downstream of
109
insulin-like growth factor-1/phosphatidylinositol-3-kinase (Igf1/PI3K) axis, induces
110
attenuation of DNA binding and its translocation to the cytoplasm from the nucleus,
111
leading to a decrease in the expression of Foxo-target genes (45, 51). Besides
112
phosphorylation, Foxo proteins are also regulated through other post-translational
113
modifications including acetylation (4, 49). By using genetic tools Bertaggia et al. (4)
114
showed that Foxo3 is progressively acetylated, which induces its cytosolic
115
localization and lower stability, during denervation and concomitantly Atrogin-1 is
116
downregulated. In addition to inhibit proteolysis, Akt also promotes protein synthesis
117
by recruiting several effectors such as mechanistic target of rapamycin (mTOR) and
118
glycogen synthase kinase 3β (GSK3β) (47).
119
The purpose of this study was to examine the morphological and molecular
120
mechanisms involved in immobilization-induced atrophy in slow-twitch soleus
121
muscles from female rats at different stages of postnatal development. Additionally,
122
we aimed at investigating the role of autophagy in regulating muscle plasticity during
123
immobilization. Our data show that muscle disorders induced by 10-day-
124
immobilization occurs in age-independent and -dependent manners. Furthermore,
6 125
although autophagic flux is accelerated following disuse in weanling and adult rats, its
126
pharmacological blockade with colchicine (COL) markedly aggravates muscle
127
abnormalities and induces a myopathic profile. Our data also provide evidences that
128
the inhibition of autophagy induced by COL may be ineffective for treating muscle
129
atrophy following immobilization.
130 131
METHODS
132
Animals and experimental procedures
133
Female weanling (21 days old, ~60g) and adult (81 days old, ~320g) Wistar rats were
134
used in the experiments. The animals were housed in a room with a 12:12 h light-dark
135
cycle and were given free access to water and a normal chow diet. For most
136
experiments, the following four experimental groups were used: control weanling
137
(CW), 10-day-immobilized weanling (IW), control adult (CA) and 10-day-
138
immobilized adult rats (IA). The duration of immobilization was chosen because
139
muscle atrophy occurred at this time point in both age groups (2, 12). The animals
140
were submitted to immobilization (see Immobilization Model) for 10 days and then
141
euthanized in a carbon dioxide chamber. Soleus muscles were removed, rolled in
142
talcum powder, frozen in liquid nitrogen, and stored at -80ºC until further processing
143
for the analysis of fiber cross-sectional area (FCSA) and immunofluorescence. For
144
determination of mRNA (qPCR) and protein (western blot) levels of the markers of
145
protein metabolism and signaling pathways, talcum powder was not used for freezing
146
muscles. We chose to analyze soleus muscle, because previous studies (2, 7, 36)
147
indicate that slow-twitch muscles like soleus undergo rapid and more pronounced
148
atrophy than fast-twitch muscles in rodent models of muscle disuse. For the
149
evaluation of autophagy flux in vivo, adult rats were treated or not with colchicine
7 150
(COL; C9754; Sigma-Aldrich) at a dose of 0.4 mg/kg/day (ip) in the last three days of
151
the immobilization (i.e., 8, 9 and 10 day). This protocol was originally proposed by Ju
152
et al. (20) and modified by us by giving an additional injection of COL 1 h prior the
153
euthanasia. Control groups received an equal volume of water (vehicle). In order to
154
induce a chronic blockade of autophagy, weanling and adult rats were concomitantly
155
submitted to immobilization and treatment with COL for 10 days. All experiments
156
and protocols were performed in accordance with the ethical principles of animal
157
research adopted by the Brazilian College of Animal Experimentation (COBEA) and
158
were approved by Ribeirão Preto Medical School of the University of São Paulo –
159
Ethical Commission of Ethics in Animal Research (CETEA; no 146/2012).
160 161
Immobilization model
162
Before the immobilization procedure, rats were anesthetized using an intraperitoneal
163
injection of 4% chloral hydrate (4 ml/kg). The right hind limb was immobilized as
164
previously described (2). Briefly, the upper part of the immobilization device is
165
similar to a T-shirt made of viscolycra, which allows free movements of the head and
166
forelimbs of the rat. The lower part of the device, divided into anterior and posterior
167
sections, consisted of a stainless steel mesh with the margins wrapped with
168
impermeable surgical tape. The anterior section was also wrapped with cotton lining
169
to protect the anterior surfaces of the immobilized limb and hip. After that, the upper
170
and lower portions of the device were joined with staples. This immobilization model
171
allows daily adaptation according to the growth of the animals during immobilization
172
and consists of maintaining the tibial-tarsal joint in maximum plantar flexion for a
173
period of 10 consecutive days.
174
8 175
Muscle preparation for histology
176
Muscles were cut into 5-µm-thick transverse sections with a Leica CM1850 UV
177
cryostat at -25°C (Leica Microsystems, Wetzlar, Germany); tissue sections were then
178
placed on 26×76 mm slides and were used for hematoxylin and eosin (H&E) and
179
Picrosirius stainings, myofibrillar adenosine tri-phosphatase (mATPase) reaction and
180
immunohistochemistry.
181 182
Histological and morphometric analysis
183
The tissue sections were stained with hematoxylin and eosin (H&E) and the images
184
were captured using an optical microscope (Leica DM 2500). Fiber cross-sectional
185
area (FCSA, expressed in µm2) was calculated by counting five random fields (X400
186
magnification) of each muscle using ImageJ software (version 1.45s, National
187
Institutes of Health, USA).
188
The analysis of the connective tissue area was performed in slides stained by
189
Picrosirius. The images were captured by a high-resolution camera (Leica DFC290,
190
Leica Microsystems, Frankfurt, Germany) connected to an optical microscope (Leica
191
DMR 2500) coupled to a polarizing filter showing the birefringence of collagen fibers
192
(X200 magnification). The quantification of connective tissue was determined by
193
counting mean area of three random fields of each muscle using the Leica QWin V3
194
software.
195
As shown in Figures 1K-N, four fiber types (I, IIA, IIC and IIX) were determined by
196
mATPase (E.C.2.1.3.5.7.9.1) histochemistry reaction (19) in acid and alkaline
197
medium (pH 4.35, 4.65 and 9.8). For the analysis of fiber type distribution and FCSA,
198
the number of each fiber type and myofiber size were determined by counting three
199
random fields of each muscle (X200 and X400 magnification, respectively, for adult
9 200
and weanling rats) using LAS software (Leica Microsystem) as described previously
201
(11). The images were captured by Leica DM 2500 light microscope.
202 203
Immunohistochemistry and fluorescence microscopy
204
A single immunofluorescence analysis of myosin heavy chain (MHC) isoforms was
205
performed with primary antibodies against MHCI (1:40) and MHCIIA (1:5). The
206
secondary antibodies used were goat anti-mouse-Red Alexa Fluor 568 (against MHC
207
I; 1:250) and goat anti-mouse IgG - Blue Alexa Fluor 350 (against MHC IIA; 1:250).
208
Muscle slices were fixed in Xpress molecular fixative (Sakura Finetek, Alphen aan
209
den Rijn, Netherlands), washed with phosphate-buffered saline (PBS) and then
210
blocked with 10% normal goat serum (Vector Laboratories, Road Burlingame,
211
California, USA) in PBS. The slices were blocked with Avidin (Avidin/Biotin
212
Blocking Kit, Vector Laboratories, Road Burlingame, California, USA), washed with
213
PBS, and incubated with the primary antibody for 2h at 37°C. After washing with
214
PBS, the slices were incubated with a secondary antibody, washed with PBS and
215
mounted with Prolong Gold Antifade without DAPI (Invitrogen). For the analysis of
216
FCSA, myofiber size was determined by counting three random fields (X200
217
magnification) of each muscle using ImageJ software.
218 219
Western blotting analysis
220
Soleus muscles were homogenized in 50 mM Tris–HCl buffer (pH 7.4) containing
221
150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS,
222
10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM sodium
223
orthovanadate, 5 µg/ml of aprotinin, 1 mg/ml of leupeptin, and 1 mM phenylmethyl-
224
sulfonyl fluoride (PMSF) at 4°C. The homogenate was centrifuged at 21,000xg at 4°C
10 225
for 20 min, and the supernatant was retained. Protein content was determined using
226
BSA as a standard (26). An equal volume of sample buffer (20% glycerol, 125 mM
227
Tris–HCl, 4% SDS, 100 mM dithiothreitol, 0.02% bromophenol blue, pH 6.8) was
228
added to the supernatant, and the mixture was boiled. Thirty to fifty micrograms of
229
total protein was separated by SDS-PAGE, transferred to nitrocellulose membranes,
230
and blotted with anti-LC3-I/II (1:1,000), anti-p62 (1:1,000), anti-atrogin-1 (1:750),
231
anti-MuRF-1 (1:750), anti-calpastatin (1:1,000), anti-m-calpain (1:750), anti-µ-
232
calpain (1:750), anti-S6 (1:1,000), anti-eIF4E (1:1,000), anti-4EBP1(1:1,000), anti-
233
GSK3β (1:1,000), anti-Ubiquitin (1:1,500), anti-20S proteasome β1 subunit (1:750),
234
anti-20S proteasome β3 subunit (1:1,000), anti-20S proteasome β5 subunit (1:750),
235
anti-Akt (1:750), anti-Foxo3a (1:750), anti-Foxo1 (1:1,000), anti-Foxo4 (1:1,000),
236
anti-phospho (p)-[Ser473]-Akt (1:500), anti-p-[Thr308]-Akt (1:500), anti-p-[Ser9/21]-
237
GSK3β/α (1:1,000), anti-p-[Ser235/236]-S6 (1:1,000), anti-p-[Thr70]-4EBP1(1:1,000),
238
anti-p-[Ser209]-eIF4E (1:1,000), anti-p-[Ser256]Foxo1 (1:500), anti-p-[Ser193]Foxo4
239
(1:500),
240
[Lys259/262/271]Foxo1 (1:250) and β-actin (1:1,000). Primary antibodies (Ab) were
241
detected using peroxidase-conjugated secondary Ab (1:1,000 for anti-p-[Ser473]-Akt,
242
anti-p-[Thr308]-Akt, anti-Akt, anti-p-[Ser193]Foxo4, anti-ac-[Lys259/262/271]Foxo1, anti-
243
p-[Thr24]Foxo1/[Thr32]Foxo3a, anti-Foxo3a, anti-Foxo4, anti-Foxo1, anti-S6, anti-
244
eIF4E, anti-4EBP1, anti-GSK3β,
245
anti-m-calpain,
246
[Ser235/236]-S6, anti-p-[Thr70]-4EBP1, anti-p-[Ser209]-eIF4E, anti-atrogin-1, anti-
247
MuRF-1, 1:5,000 for β-actin and 1:10,000 for anti-Ubiquitin, anti-20S proteasome β1
248
subunit, anti-20S proteasome β3 subunit, anti-20S proteasome β5 subunit ) and
anti-p-[Thr24]Foxo1/[Thr32]Foxo3a
anti-µ-calpain,
(1:750),
anti-acetyl
(ac)-
anti-p-[Ser256]Foxo1, anti-p62, anti-calpastatin, anti-LC3-I/II,
anti-p-[Ser9/21]-GSK3β/α,
anti-p-
11 249
visualized using ECL reagents by a Molecular Imager ChemiDoc XRS (BioRad).
250
Band intensities were quantified using Image Lab software (version 5.2.1, BioRad).
251 252
Quantitative PCR
253
Soleus muscles were harvested and immediately frozen in liquid nitrogen. RNA was
254
subsequently isolated from individual skeletal muscles using Trizol (Invitrogen).
255
Reverse transcription of RNA to cDNA was performed using 1 µg of total cellular
256
RNA, 20 pmol of oligo(dT) primer (Invitrogen), and Advantage ImProm-II reverse
257
transcriptase (Promega, Madison, WI). Quantitative (real-time) PCR was performed
258
using an ABI7000 sequence detection system (Applied Biosystems, Foster City, CA),
259
a SuperScript III Platinum SYBR Green One-Step RT-qPCR Kit with ROX
260
(Invitrogen), and primers for rat Igf1 (forward 5’-GCT TGC TCA CCT TTA CCA
261
GC-3’ and reverse 5’-AAT GTA CTT CCT TCT GGG TCT-3’) and Rpl39 (forward
262
5’-CAA AAT CGC CCT ATT CCT CA and reverse 5’-AGA CCC AGC TTC GTT
263
CTC CT). The relative quantification of mRNA levels was plotted as the fold-increase
264
in comparison to the values of the respective control group. Igf1 transcript was
265
normalized to Rpl39 levels, and the mRNA levels were calculated using the standard
266
curve method (9).
267 268
Antibodies, drugs and reagents
269
Rabbit
270
calpastatin, anti-m-calpain, anti-µ-calpain, anti-LC3-I/II, anti-p-[Ser9/21]-GSK3β/α
271
(1:1,000), anti-p-[Ser240/244]-S6, anti-p-[Thr70]-4EBP1 and anti-p-[Ser209]-eIF4E were
272
purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti-atrogin-1 and
273
anti-MuRF1 and mouse anti-β-actin were obtained from Santa Cruz Biotecnology
polyclonal
anti-p-[Ser473]-Akt,
anti-p-[Ser256]-Foxo1,
anti-p62,
anti-
12 274
(Santa Cruz, CA). The immunofluorescence Ab MHCI (#MAB1628) was purchased
275
from Merck Millipore (Darmstadt, Germany) and MHCIIA (#SC-71, 2F7) were from
276
the Developmental Studies Hybridoma Bank (University of Iowa, Iowa). The
277
secondary Abs for immunofluorescence were from Invitrogen (Life Technologies,
278
Carlsbad, CA). All other drugs and reagents were purchased from Sigma-Aldrich (St.
279
Louis, MO), Thermo Scientific HyClone (Pittsburgh, PA), Invitrogen (Carlsbad, CA),
280
Calbiochem EMD Biosciences (La Jolla, CA), or Amersham Biosciences
281
(Piscataway, NJ).
282 283
Image editing
284
Histological and western blot images were cropped and labeled and adjustments in
285
brightness and contrast were applied to the entire figure in Adobe Photoshop CS4
286
Extended (Adobe) and Microsoft PowerPoint (Microsoft).
287 288
Statistical analysis
289
The data are presented as the mean ± standard error (SE). The means from different
290
groups were analyzed using unpaired Student’s t-tests or Mann–Whitney rank test for
291
the non-parametric samples. Multiple comparisons were made using one-way or two-
292
way ANOVA followed by a Holm-Sidak post hoc test. P ≤ 0.05 was taken as the
293
criterion for significance.
294 295
RESULTS
296
Immobilization induces skeletal muscle atrophy and fiber type transition in weanling
297
and adult rats
13 298
Figure 1 displays the wet mass, the ratio of muscle mass-to-body mass, and the fiber
299
cross-sectional area (FCSA) of the normal control or 10-day-immobilized solei in
300
female rats at different stages of postnatal development. Immobilization significantly
301
reduced the wet mass [71% in weanling (IW) and 55% in adult (IA) rats] and the ratio
302
of muscle mass-to-body mass (44% in IW and 45% in IA) of solei. H&E stained
303
fibers confirmed muscle atrophy by the reduction in FCSA in both IW (53%) and IA
304
(22%) groups (data not shown). Although both age groups also presented small
305
flattened fibers after immobilization (Figure 1), only IW rats showed small groups of
306
atrophic fibers (Figure 1E) and IA rats presented a few target fibers, with central
307
basophilia surrounded by a condensed zone (Figure 1H). Mammalian skeletal muscles
308
contain multiple fiber types that can be classified based on four major myosin heavy
309
chain (MHC) isoforms; one slow isoform (MHCI) and three fast isoforms [MHCIIA,
310
MHCIIX, MHCIIB) (48). The coexpression of MHCI and MHCIIA isoforms results
311
in the formation of hybrid type IIC fibers (39). Consistent with H&E staining, the
312
evaluation of the MHC isoforms by myosin adenosine triphosphatase (mATPase)
313
histochemistry revealed that immobilization reduced the FCSA of types I (62%), IIA
314
(95%), IIC (47%) and IIX (58%) in IW group and types I and IIC (~45%) in IA group
315
(Figures
316
independently of age, as indicated by the reduction in the quantity of type I fibers
317
(~35%) in IW and IA groups and the increase in types IIA (~50% in both groups), IIC
318
(88% in IA) and IIX (64% in IW) fibers (Figures 1K-P).
1I-N).
Immobilization
also
induced
slow-to-fast
fiber
switching
319 320
Immobilization reduces markers of protein synthesis in skeletal muscle from weanling
321
and adult rats
14 322
mTOR regulates numerous components involved in the synthetic machinery, although
323
mTOR-independent pathways may also contribute to protein synthesis and muscle
324
growth (34, 41). The activation of mTOR leads to phosphorylation and activation of
325
p70S6k (also known as S6K), which, in turn, phosphorylates the ribosomal protein
326
S6. mTOR also inhibits the eukaryotic initiation factor 4E (eIF4E) binding protein 1
327
(4EBP1), which inhibits 5′-cap-dependent mRNA translation by binding and
328
inactivating eIF4E. GSK3α/β isoforms act as repressors of the eukaryotic initiation
329
factor 2B (eIF2B), reducing protein synthesis at the initiation step in an mTOR-
330
independent manner. Immobilization induced a marked reduction in the
331
phosphorylation levels of the ribosomal protein S6 (74% in IW and 57% in IA) and
332
GSK3β (44% in IW and 28% in IA) in soleus muscles. GSK3α phosphorylation was
333
unaltered in atrophied solei from IA and IW rats (Figures 2D). The phosphorylation
334
status of eIF4E, a posttranslational modification that negatively regulates its
335
interaction with 4EBP1, was slightly increased (~10%) only in muscles from IA
336
(Figure 2C). The protein content of S6 and eIF4E was downregulated (31%) in IA and
337
upregulated (67%) in IW, respectively. In addition, the total content of 4EBP1 were
338
higher (~50%) in both age groups compared with control muscles, however, its
339
phosphorylated form did not change in any group. Thus, the dephosphorylation of S6
340
and GSK3 and the upregulation of 4EBP1 suggest an impairment of protein synthesis,
341
which may contribute to muscle wasting in immobilized muscles from weanling and
342
adult rats.
343 344
Autophagy is a common proteolytic system stimulated in immobilized muscles from
345
weanling and adult rats
15 346
Because the rapid loss of muscle mass in a variety of pathological conditions
347
primarily results from accelerated protein breakdown (10, 15), we evaluated the
348
protein content of different components of the proteolytic systems in immobilized
349
solei from weanling and adult rats. As previously reported, autolytic cleavage of the
350
large subunit of calpains is correlated with the reduction in Ca2+ concentration
351
required for proteolytic activity and can be used as a marker of calpain activation
352
(25). Despite the fact that neither the full-length (80-kDa) nor the total content of μ-
353
calpain was changed by immobilization, its autolyzed form (75-kDa) was highly
354
increased (45%) in muscles from IW rats (Figures 3A-C). In adult rats,
355
immobilization did not cause any effect on μ-calpain autolysis, but there was a
356
tendency toward increased levels of the full-length and the total content of this
357
protease, which did not reach statistical significance (P=0.08). Note that only the full-
358
length form of μ-calpain is detected in basal conditions. Neither m-calpain nor
359
calpastatin was altered in immobilized muscles from any group (Figures 3A, D and
360
E). Figures 4A-B shows that immobilization induced a significant increase in high
361
molecular weight (65-130kDa) ubiquitinated proteins (2-fold) and the protein content
362
of the Ub-ligase MuRF1 (90%), but not Atrogin-1, in muscle from IA group. No
363
changes were detected in the ubiquitinated proteins and the protein levels of these Ub-
364
ligases in IW rats (Figures 4C-D). In additional, the protein level of the catalytic (β1
365
and β5) and structural (β3) subunits of 20S proteasome did not change in any group
366
(Figures 4C and E). Autophagy is usually evaluated by the level of the LC3 forms and
367
p62 (22). ProLC3 is processed to its cytosolic form, LC3-I, and modified to a
368
membrane-bound
369
autophagosomes. The adaptor protein SQSTM1/p62, in turn, mediates the delivery of
370
ubiquitinated protein aggregates to ALS. Furthermore, p62 was identified as one of
form,
LC3-II,
that
localizes
to
preautophagosomes
and
16 371
the specific substrates that are degraded through ALS. By analyzing the conversion of
372
LC3-I to LC3-II, we observed that LC3-II content (~3-fold in both age groups) and
373
the LC3-II/LC3-I ratio (~4-fold in IW and ~2-fold in IA) were upregulated in
374
immobilized solei (Figures 5A, and B). Intriguingly, p62 protein content was reduced
375
and increased, respectively, in IW (80%) and IA (2.5-fold) (Figures 5A and C),
376
suggesting that autophagy is enhanced in immobilized muscles from weanling rats but
377
our results are inconclusive in adult rats. Because LC3-II and p62 are degraded by
378
lysosomal hydrolytic enzymes, the measurement of “static” levels of these proteins
379
may cause possible misinterpretations (22). To further investigate the autophagic flux
380
in adult rats, we evaluated LC3-II protein content in soleus from animals that were
381
treated or not with colchicine (COL; 0.4 mg/kg/day), a lysosomotropic agent that
382
blocks the degradation of LC3-II and p62, in the last three days of immobilization. In
383
IA rats, COL treatment exacerbated the increase by 98% in the protein levels of LC3-
384
II, without altering the effects of immobilization on the content of p62 and MuRF1
385
(Figures 5D-F). Taken together, these findings show that immobilization-induced
386
muscle atrophy stimulates different proteolytic systems depending on the stage of
387
rat’s postnatal development. However, autophagic flux is enhanced in immobilized
388
muscles from weanling and adult rats, suggesting that ALS may be necessary for the
389
maladaptations induced by muscle inactivity.
390 391
Chronic inhibition of autophagy aggravates immobilization-induced muscle atrophy
392
We next monitored the relevance of chronic blockade of autophagy in atrophying
393
muscle. For that, adult rats were concomitantly submitted to immobilization and
394
treatment with COL for 10 days. COL treatment led to a further reduction in muscle
395
mass (28%) and FCSA (40%; data not shown) in H&E stained myofibers and a huge
17 396
elevation in the area of connective tissue (7-fold) surrounding myofibers in
397
immobilized soleus (Figure 6). It is noteworthy that H&E staining in immobilized
398
muscles
399
degeneration-regeneration process and myopathy such as central nuclei, small
400
flattened or irregularly shaped atrophic fibers, several target fibers and amorphous
401
materials like necrotic fiber (Figures 6B-E). In addition, immunofluorescence analysis
402
of MHC expression showed a further decrease (37%) in FCSA of type I fibers in IA
403
rats treated with COL (Figure 6Q). In spite of no effect of immobilization, FCSA of
404
type IIA fibers in muscles from immobilized rats treated with COL was smaller (44%)
405
than control group (Figure 6Q). In contrast to our initial hypothesis, these data
406
indicate that the inhibition of autophagy system during immobilization induces
407
muscle degeneration and more severe atrophy. Weanling rats were also treated with
408
COL but the dose was lethal and all animals died within a few days. Further
409
experiments are needed to determine the safety and tolerability of the dose capable of
410
blocking autophagy in young female rats.
from
COL-treated
rats
showed
morphological
characteristics
of
411 412
Immobilization deactivates Igf1/Akt signaling in skeletal muscle from weanling and
413
adult rats
414
The growth factor Igf1 and its intracellular mediator Akt are crucial regulators of
415
protein metabolism promoting muscle hypertrophy (41). Besides, Akt can
416
phosphorylate and inhibit the master regulators of muscle atrophy, Foxo transcription
417
factors (45, 51). Therefore, we assessed the gene expression of Igf1 and the
418
phosphorylation status of Akt and Foxo1, 3 and 4 in immobilized solei from weanling
419
and adult rats. Immobilization caused a downregulation in the mRNA levels of
420
muscle Igf1 in both IW (52%) and IA (65%) groups, which was paralleled by a
18 421
significant reduction in the phosphorylation levels of Ser473-Akt (~50% in both ages)
422
and Thr308-Akt (~70% in IW and 30% in IA) (Figures 8A-D). In spite of the
423
downregulation in Igf1/Akt signaling, the phosphorylation levels of Foxo1 (~8-fold in
424
Ser256 and ~5-fold in Thr24) and Foxo4 (2-fold), normalized to the respective total
425
proteins, were increased only in muscles from IW group (Figures 8B-E). However,
426
analyzing the ratio of phosphoprotein to the loading control β-actin, we observed that
427
the phosphorylation levels in different residues of Foxo1, 3 and 4 were not changed in
428
any group (data not shown). Interestingly, the acetylation levels of Foxo1 were
429
increased (~20-fold) and the protein content of Foxo1 and 4 were decreased by 90%
430
and 48%, respectively, in solei from only IW group (Figures 8B-C and E-F).
431
Altogether, these findings suggest that the deactivation of Igf1/Akt signaling pathway
432
may be responsible for the inhibition of markers of protein synthesis and the
433
stimulation of the proteolytic systems, especially autophagy, in muscles from rats at
434
different stages of postnatal development. In addition, Foxo1 and 4 downregulation in
435
muscles from 10-day-immobilized weanling rats may represent an intrinsic muscle
436
mechanism to prevent excessive tissue loss.
437 438
DISCUSSION
439
Traumatic orthopedic injury is a major public health problem in both childhood and
440
adulthood (16, 32). Following an injury, reduced muscle activity secondary to
441
immobilization induces fiber type transition, muscle wasting and weakness (7, 31,
442
42). Although these maladaptations in adult individuals are well documented (7), to
443
our knowledge, there is no information in the literature comparing cellular and
444
molecular mechanisms involved in such effects during immobilization in weanling
445
and adult rodents. In addition, only a few studies have investigated the atrophic
19 446
response of infant muscles, which is an important step toward developing an efficient
447
therapy in children and improving the rehabilitation process.
448
This study demonstrates that, despite the differences in the effect size, immobilization
449
for 10 days resulted in slow-to-fast fiber transition and atrophy in rat soleus muscle in
450
weanling and adult female rats. In accord with previous studies (3, 12), we observed a
451
reduced proportion of type I fibers and an increased proportion of type IIA fibers in
452
immobilized solei in an age-independent manner. Additionally, the quantity of type
453
IIX and IIC fibers was also increased in muscles from IW and IA groups,
454
respectively. Because type I and IIA fibers exhibit high oxidative potential and
455
resistance to fatigue and slow contraction time, and type IIX/D and IIB fibers are
456
primarily glycolytic and have faster contraction time and rapid fatigue profile (48),
457
the slow-to-fast fiber shift could probably be responsible for the reduction of the
458
fatigue resistance observed in adult patients following immobilization (50). However,
459
muscle weakness and dysfunction following immobilization are thought to be a
460
consequence of muscle atrophy (31, 42). As expected, our data show that the mass
461
and FCSA of slow-twitch soleus muscles following disuse were smaller than control
462
muscles. Moreover, the reduction in FCSA measured in H&E stained fibers was more
463
pronounced in weanling rats than in adult ones, which seems to be related to a
464
significant decrease in the area of type IIA and IIX fibers observed only in
465
immobilized solei from weanling rats supporting the idea that muscles of animals in
466
the growing stage are more sensitive to atrophy. The available data do not allow for a
467
conclusion to be reached regarding the reason for the differences between young and
468
adult rats, but it seems reasonable to speculate that muscle inactivity may have caused
469
a downregulation of the local growth factors required to induce muscle growth, such
470
as Igf1 and mechano growth factors. Indeed, we observed that immobilization caused
20 471
a downregulation of Igf1 mRNA in both age groups. However, it has been shown that
472
weanling (3-week-old) transgenic mice expressing a dominant negative IGF-I
473
receptor specifically in skeletal muscle had muscle hypoplasia and a more severe
474
muscle atrophy when compared with adult (8-week-old) transgenic mice (14),
475
suggesting that growth factors may cause more pronounced effects on muscle mass
476
during childhood.
477
Skeletal muscle mass is maintained by a delicate balance between protein synthesis
478
and protein degradation. Attenuated rates of muscle protein synthesis have been
479
observed in both human (17) and animal (21) models of disuse atrophy. Igf1 is an
480
anabolic growth factor that is crucial for regulating protein metabolism and muscle
481
growth (14, 41, 51). Akt, in turn, is a central intracellular mediator of Igf1 that
482
promotes muscle anabolic effects (41) by phosphorylating a number of proteins (6,
483
41). In agreement with previous findings (6), our model of muscle disuse in weanling
484
and adult rats also downregulated Akt phosphorylation. As a consequence, the
485
phosphorylation levels of ribosomal protein S6 and GSK3β were reduced in the same
486
muscles, indicating an inhibition and stimulation, respectively, of the activity of these
487
proteins. S6 protein is a good readout for Akt/mTOR/S6K signaling pathway because
488
Akt activates mTOR that leads to the activation of S6K, which, in turn, directly
489
phosphorylates S6. The importance of S6K in muscle physiology has been shown in
490
knockout animals that present muscle atrophy (35). GSK3β, however, induces muscle
491
loss not only by inhibiting protein synthesis but also by stimulating protein
492
breakdown (56). Therefore, our data suggest that the molecular mechanism involved
493
in the suppression of protein synthesis by immobilization in both weanling and adult
494
rats depends, at least in part, on the downregulation of Igf1 signaling, especially
495
Akt/GSK3 and Akt/mTOR/S6K/S6 pathways.
21 496
On the other hand, previous studies have reported that muscle atrophy in a variety of
497
pathophysiological settings is mainly due to an acceleration of the rate of protein
498
degradation (7, 15, 23). Multiple proteolytic systems control protein turnover in
499
skeletal muscle cells. Our data indicate that Ca2+-dependent proteolysis is enhanced in
500
immobilized muscles from weanling rats, but not from adult ones. However, this
501
finding in adult rats does not rule out the possibility that an increase in the markers of
502
Ca2+-dependent proteolysis and/or in its proteolytic activity occurred prior to the end
503
of the 10-day immobilization, which was the only time interval examined in the
504
present study. Indeed, Talbert et al. (52) have previously demonstrated that casting
505
significantly increased the content of the autolyzed form of μ-calpain following 7
506
days of immobilization in adult rats. In addition, pharmacological or genetic
507
inhibition of calpains with SJA-6017 (52) or by overexpressing calpastatin in
508
transgenic mice (53), respectively, has been able to attenuate inactivity-induced
509
muscle wasting in adult rodents, indicating that Ca2+-dependent system participates in
510
the induction of muscle atrophy in both age groups.
511
Nevertheless, the deactivation of Igf1/Akt signaling by immobilization might also
512
result in the downregulation of Foxo phosphorylation and an increase in its
513
transcriptional activity (44, 51). Because Foxo transcription factors coordinate the
514
activation of the UPS and ALS in atrophying muscles (28, 30, 46, 51), we evaluated
515
the content of specific readouts for these proteolytic systems. The content of
516
ubiquitinated proteins and the atrophy-related Ub-ligase MuRF1, but not Atrogin-1,
517
was increased only in immobilized adult muscles. Similar results were obtained by
518
Madaro et al. (27) in the mRNA levels of MuRF1 and Atrogin-1 in tibialis anterior
519
muscle from adult mice immobilized for 7 days. Unexpectedly, the phosphorylation
520
levels of Foxo3 were not altered by immobilization in any group. More interestingly,
22 521
the phosphorylation levels and the content of Foxo1 and 4 were up- and down-
522
regulated, respectively, in muscles from IW rats, which could explain the fact that
523
neither Atrogin-1 nor MuRF1 were induced in this age group. However, it seems
524
highly likely that Foxo dephosphorylation and Ub-ligases upregulation would have
525
occurred in an earlier time interval, i.e., long before the muscles were sampled at 10
526
days after immobilization. This hypothesis is supported by some findings: 1) the
527
mRNA expression of both MuRF1 and Atrogin-1 peaks at 3 days after immobilization
528
(5, 23) and 2) Foxo3 content and Atrogin-1 expression were progressively
529
downregulated during sciatic denervation (4). Additionally, the acetylation of Foxo1,
530
3 and 4 by acetyltransferase p300 prevents their activation (4, 49). In our models,
531
Foxo1 was hyperacetylated in 10-day-immobilized muscles from weanling rats,
532
similarly to Foxo3a in 14-day-denervated muscles from adult mice as reported by
533
Bertaggia et al. (4). Because other post-translational modifications that regulate Foxo
534
activity and the intracellular localization of Foxo were not evaluated, it is also
535
possible that at least one member of Foxo family was still active in our models but we
536
were unable to determined. Lastly, despite no change being observed in the content of
537
proteasome subunits in our experiments, the requirement of UPS for disuse-induced
538
muscle loss has been reported previously (1, 5, 23). For instance, it has been shown
539
that the pharmacological blockade of proteasome or the genetic deletion of MuRF1 is
540
able to attenuate muscle loss by immobilization or denervation in adult animals.
541
Among the proteolytic systems, autophagy was the only degradative pathway
542
regulated in both age groups 10 days after immobilization suggesting that ALS could
543
be required for the maintenance of muscle atrophy. In muscles from weanling rats,
544
immobilization caused the conversion of the cytosolic form LC3 (LC3-I) to the
545
autophagosome-bound form LC3 (LC3-II) and reduced p62 protein content,
23 546
indicating an accelerated autophagic flux. An expected pattern is that the protein
547
levels of p62 vary according to the autophagic flux, i.e., low p62 content is seen
548
during autophagy activation and the accumulation of p62 protein correlates with
549
autophagy inhibition (22). On the other hand, the synthesis of p62 can increase during
550
autophagy to replenish p62 protein degraded in ALS (38). Similar to p62, LC3-II can
551
increase, decrease or even remain unchanged in the setting of autophagic induction
552
because LC3-II is both produced and degraded during autophagy (22). Indeed, in
553
muscles from adult rats, immobilization also resulted in the LC3-I to LC3-II
554
conversion, but instead of decreasing p62 content, this protein accumulated in
555
atrophied muscles, suggesting an impairment of autophagic flux. To avoid any
556
misinterpretation, an “autophagometer” technique (20) was applied by measuring the
557
content of LC3 forms and p62 in muscles from 10-day-immobilized rats. For that,
558
adult rats were treated or not for the last three days with the lysosomotropic agent
559
COL, which destabilizes microtubules impairing autophagosome-lysosome fusion.
560
Despite no additional changes in p62 content, the acute blockade of autophagy by
561
COL induced a further increase in LC3-II content in immobilized muscles.
562
Altogether, these findings suggest that immobilization-induced atrophy may be in part
563
due to enhanced autophagy in muscles from both weanling and adult rats.
564
Because limiting autophagy by haploinsufficiency of the autophagy gene Beclin-1
565
diminishes muscle atrophy and maintains motor function in a mouse model of spinal
566
and bulbar muscular atrophy (58), and the inhibition of calpains or UPS by different
567
approaches did not completely prevent protein degradation and muscle atrophy (1, 5,
568
23, 52, 53), we asked whether COL-induced blockade of autophagy concomitant with
569
immobilization for 10 days could avoid muscle disorders in adult rats. In contrast,
570
chronic inhibition of autophagy aggravated muscle wasting in immobilized solei,
24 571
which also displayed degenerative and myopathic features with increase in the area of
572
connective tissue. This view is consistent with previous findings (8, 29, 40, 57),
573
despite being contrary to our hypothesis. COL has been primarily used for treating
574
gout and other rheumatic and nonrheumatic disorders but adverse effects have been
575
described (13, 57). Amongst them, myopathy with the accumulation of autophagic
576
vacuoles is a rare side effect that has been reported in humans and rodents (8, 57).
577
The risk of COL-induced myopathy may increase with duration of treatment and
578
medication combination. Indeed, although COL treatment for 40 days caused
579
myopathy with rare vacuoles, no evidence of myopathy was observed in mice treated
580
for a shorter period (i.e., 10 days) (8). Interestingly, the incidence and severity of
581
COL-induced muscle toxicity may be markedly increased by concomitant treatment
582
with autophagy inducers (e.g., rapamycin and statins) (8). Thus, it is likely that
583
immobilization-induced autophagy may have triggered and hastened the development
584
of the COL myopathy.
585
It is also interesting to note that target fibers were rarely seen in disused muscles from
586
adult rats but were highly present after autophagy blockade. Target fibers have been
587
described as myofibers in an active catabolic state that accumulates autophagic
588
multivesicular bodies at the center of the cell (33). Additionally, mitochondrial
589
swelling and disintegration was detected in the condensed zone surrounding the target
590
center. To our knowledge, this kind of abnormality (i.e., target fiber) has never been
591
reported as a feature of COL-induced myotoxicity in both normal and disused
592
muscles. Nevertheless, it has been recently shown the accumulation of the vacuolated
593
material in the center of myofibers, a characteristic of target fiber, from a patient
594
concurrently treated with COL and a statin (8). The accumulation of autophagic
595
substrates, such as potentially toxic ubiquitinated proteins and abnormal
25 596
mitochondria, has been associated with a disarrangement of sarcomeres (29) and
597
microtubules (40), muscle weakness (29) and atrophy (29, 40) in two different strains
598
of
599
haploinsufficiency mice, Atg7 or Atg5 homozygous knockout mice, which display an
600
impairment in autophagy, showed an exacerbation of the muscle loss during
601
denervation and fasting (29) and the myopathic phenotype in a mouse model of
602
Pompe disease (40). Therefore, it seems reasonable to suggest that the accumulation
603
of damaged proteins and altered organelles by autophagy blockade could have
604
triggered degenerative processes and contributed to worsen muscle atrophy in
605
immobilized muscles. Further experiments are needed to confirm this hypothesis and
606
to identify the autophagic substrates that should be removed under disuse conditions.
607
Our results are also in accordance with the idea proposed by Sandri et al. (43) in
608
which the excessive activation or the strong inhibition of proteolysis may contribute
609
to muscle degeneration and thus be detrimental during pathological conditions.
610
Therefore, we may not rule out that autophagy inhibitors may be used for treating
611
muscle wasting, however, other drugs that can specifically target different control
612
points in autophagy system should be tested and the exact therapeutic dose of these
613
agents need to be optimized according to the disease.
614
In summary, the data in the present work demonstrate that although unilateral hind
615
limb immobilization for 10 days induces deactivation of Igf1/Akt signaling and
616
markers of protein synthesis, enhancement of autophagy flux, slow-to-fast fiber
617
switching and muscle atrophy in an age-independent manner, the regulation of Foxo
618
factors and the stimulation of UPS and Ca2+-dependent proteolytic systems varies
619
according the stage of postnatal development. These findings indicate that more
620
attention should be given to determine the cellular and molecular mechanisms of
muscle-specific autophagy-deficient
mice. In addition, unlike Beclin-1
26 621
atrophic response in infants in order to develop an efficient therapy for this age group.
622
Finally, pharmacological inhibition of autophagy exacerbates muscle disorders
623
induced by immobilization causing a myopathic profile.
624 625
ACKNOWLEDGMENTS
626
We thank Prof. Dr. Luciano Neder for providing laboratory facilities for this work and
627
Deise Lúcia Chesca, Cibele Prado and Keite Noguti for their technical assistance at
628
Department of Pathology, FMRP-USP. We are also indebted to Lilian do Carmo
629
Heck, Neusa Maria Zanon, Elza Aparecida Filippin, Maria Antonieta R. Garófalo,
630
and Victor Diaz Galban for their technical assistance at Departments of Physiology
631
and Biochemistry & Immunology, FMRP-USP.
632 633
GRANTS
634
This work was supported by grants from the Fundação de Amparo à Pesquisa do
635
Estado de São Paulo (FAPESP 13/08553-9 to S.P.G., 10/11015-0 to F.A.G.,
636
12/24524-6 to I.C.K. and 12/18861-0 to D.A.P.G.), from the Coordenação de
637
Aperfeiçoamento de Pessoal de Nível Superior (CAPES 373886/2014-4 to C.S.F. and
638
PNPD20131672 to W.A.S.), from the Fundação de Apoio ao Ensino, Pesquisa e
639
Assistência from the Clinics Hospital of Ribeirão Preto Medical School and Conselho
640
Nacional de Desenvolvimento Científico e Tecnológico (FAEPA 1208/2013 and
641
CNPq, respectively, to A.C.M.S.).
642 643
DISCLOSURES
644
No conflicts of interest, financial or otherwise, are reported by the authors.
645
27 646
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834
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836 837 838
LEGENDS
839 840
Fig. 1. Effects of unilateral hind limb immobilization for 10 days on the mass (A and
841
B), the histomorphological features (C-H), the fiber cross-sectional area (FCSA; I and
842
J) and the fiber type distribution (K-P) of soleus muscles from weanling and adult
843
rats. Representative images of hematoxylin and eosin (H&E) staining and mATPase
844
reaction of muscle cross sections from control weanling (CW; C and K), immobilized
845
weanling (IW; D, E and L), control adult (CA; F and M) and immobilized adult (IA;
35 846
G, H and N) rats. Scale bar, 25 μm. Arrowhead: small flattened fibers. Dashed arrow:
847
target fibers. Circle: small group of atrophic fibers. Values are presented as means ±
848
SE of 5 animals. *P ≤ 0.05, immobilized group vs. age-paired control group.
849 850
Fig. 2. Effects of unilateral hind limb immobilization for 10 days on total protein
851
content (E) and phosphorylation levels of S6 (p-Ser235/236; C), eIF4E (p-Ser209; C),
852
4EBP1 (p-Thr70; C) and GSK3β/α (p-Ser9/21; D) in soleus muscles from weanling and
853
adult rats. Membranes were stripped and reprobed for total proteins and β-actin as a
854
loading control. Phosphorylated proteins were normalized to total proteins. Total
855
proteins, in turn, were normalized to β-actin. Representative immunoblots (A and B)
856
and densitometry values (C-E) are shown. Values are presented as means ± SE of 4-7
857
animals. *P ≤ 0.05, immobilized group vs. age-paired control group.
858 859
Fig. 3. Effects of unilateral hind limb immobilization for 10 days on protein content
860
of μ-calpain (B and C), m-calpain (D) and calpastatin (E) in soleus muscles from
861
weanling and adult rats. Membranes were stripped and reprobed for β-actin as a
862
loading control. Representative immunoblots (A) and densitometry values (B-E) are
863
shown. Values are presented as means ± SE of 4 animals. *P ≤ 0.05, immobilized
864
group vs. age-paired control group. + Lysate from 7-day denervated mouse tibialis
865
anterior muscle, which was used as a positive control for the autolyzed form of μ-
866
calpain.
867 868
Fig. 4. Effects of unilateral hind limb immobilization for 10 days on protein content
869
of ubiquitinated proteins (B), Atrogin-1 (D), MuRF1 (D) and proteasome β-subunits
870
(β1, β3 and β5; E) in soleus muscles from weanling and adult rats. Membranes were
36 871
stripped and reprobed for β-actin as a loading control. Representative immunoblots
872
(A and C) and densitometry values (B, D and E) are shown. Values are presented as
873
means ± SE of 3-5 animals. *P ≤ 0.05, immobilized group vs. age-paired control
874
group.
875 876
Fig. 5. Effects of unilateral hind limb immobilization for 10 days on protein content
877
of LC3 (B and E) and p62 (C and F) in soleus muscles from weanling and adult rats
878
treated (E and F) or not (B and C) with colchicine (COL; 0.4 mg/kg/day; ip) in the
879
last three days of the immobilization. Membranes were stripped and reprobed for β-
880
actin as a loading control. Representative immunoblots (A and D) and densitometry
881
values (B, C, E and F) are shown. Values are presented as means ± SE of 3-5 animals.
882
*P ≤ 0.05, immobilized group vs. age-paired control group. # P ≤ 0.05, IA + COL
883
group vs. CA + COL group. & P ≤ 0.05, IA + COL group vs. IA group.
884 885
Fig. 6. Effects of chronic blockade of autophagy by colchicine treatment (COL; 0.4
886
mg/kg/day; ip) concomitant with immobilization for 10 days on the mass (A), the
887
histomorphological features (B-E), the area of connective tissue (J), and the fiber
888
cross-sectional area (FCSA; Q) of soleus muscles from adult rats. Representative
889
images of muscle cross sections from control (K and L) and immobilized adult rats
890
treated with saline (IA; B, F, G, M and N) or COL (IA + COL; C-E, H, I, O and P).
891
Scale bar, 25 μm. Arrowhead: small flattened fibers. Dashed arrow: target fibers.
892
Circle: small group of atrophic fibers. Dashed circle: amorphous materials like
893
necrotic fiber. χ: centrally nucleated myofiber. Values are presented as means ± SE of
894
5 animals. *P ≤ 0.05, IA + saline (IA) vs. CA. #P ≤ 0.05, IA + COL group vs. IA +
895
saline (IA). ψP ≤ 0.05, IA + COL group vs. CA.
37 896 897
Fig. 7. Effects of unilateral hind limb immobilization for 10 days on mRNA levels of
898
Igf1 (A) and total protein content (F) and phosphorylation levels of Akt (p-Ser473 and
899
p-Thr308; D), Foxo1 (p-Ser256, p-Thr24 and ac-Lys259/262/271; D and E), Foxo3 (p-Thr32;
900
D) and Foxo4 (p-Ser193; E) in soleus muscles from weanling and adult rats.
901
Membranes were stripped and reprobed for total proteins and β-actin as a loading
902
control. Phosphorylated proteins were normalized to total proteins. Total proteins, in
903
turn, were normalized to β-actin. Representative immunoblots (B and C) and
904
densitometry values (D-F) are shown. The gene expression levels of Igf1 were
905
analyzed, using Rpl39 as an endogenous control. Values are presented as means ± SE
906
of 3-5 animals. *P ≤ 0.05, immobilized group vs. age-paired control group.
0,08 0,06 0,04
*
*
0,02
IW
J
1200 900
*
600 300
*
0
* *
4000
CA
3000
*
2000
I
Fiber type
CW
C
D
IW
IW
E
*
1000
I IIA IIC IIX
0,00
IA
2
2
CW
FCSA (m )
*
0,10
1500
FCSA (m )
*
B
(g/100g of body mass)
0,30 0,25 0,20 0,15 0,10 0,05 0,00
IW IA
Soleus/body mass
Soleus mass (g)
A
CW CA
I
IIA IIC IIX Fiber type
CW
K
L
IW
IIX
I IIC IIA
IIX IIA
CA
F
G
IA
IA
H
M
CA
IIX
I
IIC
N
I
I
IIA
IIC IIA
IIX
IIC
O
100 80
Type I Type IIA
60
Type IIC Type IIX
**
40
*
20 0
CW
IW
P
% of fiber type
H % of fiber type
G
IA
100 80 60
**
40 20
*
0
CA
IA
A
B
p-Ser235/236 S6
S6
p-Ser209 eIF4E
eIF4E
p-Thr70 4EBP1
4EBP1
p-Ser21 GSK3α p-Ser 9 GSK3β
GSKβ 3 β-actin
CW CA
IW IA
* 1 BP 4E hr pT
pS
er
eI
er
F4 E
S6
**
E 1,2 1,0 0,8 0,6 0,4 0,2 0,0
* * pSer-GSK3
pSer-GSK3
Protein content/-actin ratio (relative to control)
2,4 2,0 1,6 1,2 0,8 0,4 0,0
Phosphorylated proteins (relative to control)
D
pS
Phosphorylated proteins (relative to control)
C
* * *
2,0 1,6 1,2
*
0,8 0,4 0,0
S6
eIF4E 4EBP1 GSK3
A -calpain
+
Full Lenght Autolyzed m-calpain Calpastatin
40
C
30 1,5 1,0 0,5 0,0
* Full Autolyzed lenght
Total content
E 2,0 1,5 1,0 0,5 0,0
CA
IA
50 40 30 20 10 0
2,0 1,5 1,0 0,5 0,0
N.D. N.D.
IW
Calpastatin/-actin ratio -calpain/-actin ratio (relative to control) (arbitrary units)
D
CW
N.D.
B
m-calpain/-actin ratio -calpain/-actin ratio (arbitrary unit) (relative to control)
β-actin
Full Autolyzed lenght
Total content
A
C 130 kDa
Ubiquitinated proteins
Atrogin-1
MuRF1 β1 subunit
65 kDa 130 kDa
Ponceau
β3 subunit β5 subunit β-actin
65 kDa
2.4 1.8 1.2 0.6 0.0
CW CA
IW IA
*
E 2,4 2,0 1,6 1,2 0,8 0,4 0,0
*
Atrogin-1
MuRF1
Protein content/-actin ratio (relative to control)
3.0
D Protein content/-actin ratio (relative to control)
Ubiquitinated proteins/-actin ratio (relative to control)
B
1,6 1,2 0,8 0,4 0,0
1 subunit 3 subunit 5 subunit
Protein content/-actin ratio (relative to control)
C
5
4
3 CW CA IW IA
** * *
2
1 LC3-I LC3-II LC3-II/I 3
0
*
2
1
* Protein content/-actin ratio (relative to control)
B
p62/-actin ratio (relative to control)
LC3-I LC3-II
p62 p62
β-actin
MuRF1
E F
9 8 7 6 5 4 3 2 1 CA IA CA+COL IA+COL
&#
#
LC3-I LC3-II LC3-II/I
Protein content/-actin ratio (relative to control)
A D
LC3-I LC3-II
β-actin
15
12
0
*#
9
6
3
*#
p62 MuRF1
(g/100g of body mass)
Soleus/body mass
A 0,05
IA
0,04
MHC I
IA+COL
*
0,03
MHC IIA K
L
0,02 0,01
Light microscope
0,00
B
C
Polarized microscope
F
G
M
N
H
I
O
P
c
E
D
Q
700000 600000 500000 400000 300000 200000 100000 0
*
4000
FCSA (m2)
2
Connective tissue (m )
c
J
3000 2000
CA
IA
IA + COL
*#
1000 0
I
IIA Fiber type
c
8 6
*
1,5
* E F
Protein content/-actin ratio (relative to control)
D
1
o4
xo
ox
Fo
rF
pS e
ys
30 20 10 8 6 4 2 0 -L
0.0
xo 1
*
Fo
*
er
1.5
pS
IW IA
Ac
1
Fo xo
CW CA
Phosphorylated proteins (relative to control)
hr
xo 3
** *
pT
1,0
Fo
t
0.5
hr
Ak
1.0
pT
0,0 t
0,5
hr
Ak
(relative to control)
Igf1 mRNA 2.0
pT
er
pS
Phosphorylated proteins (relative to control)
A B C
p-Ser473 Akt p-Thr308 Akt
p-Thr32 Foxo3a p-Thr24 Foxo1 p-Ser256 Foxo1 p-Ser193 Foxo4
Akt Foxo3a
Ac-Lys Foxo1
Foxo1 Foxo4 β-actin
*
* * 1,6
1,2
0,8
0,4
0,0
*
*
Akt Foxo3 Foxo1 Foxo4
