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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

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in rats at different stages of postnatal development: the role of autophagy

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Camila Silva Foresto1,4, Sílvia Paula-Gomes3,4, Wilian Assis Silveira2, Flávia

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Aparecida Graça2, Isis do Carmo Kettelhut2,3, Dawit Albieiro Pinheiro Gonçalves2,3,5,

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Ana Claudia Mattiello-Sverzut1,5

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Departments of 1Biomechanics, Medicine and Rehabilitation of the Locomotor

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Apparatus,

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School, University of São Paulo, Brazil. These authors contributed equally to this

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2

Physiology and

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Biochemistry/Immunology, Ribeirão Preto Medical

work (4first and 5senior authors).

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Correspondence to: Dawit Albieiro Pinheiro Gonçalves

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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]

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Abbreviated title: Autophagy’s role in immobilized muscle

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Copyright © 2016 by the American Physiological Society.

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ABSTRACT

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Muscle loss occurs following injury and immobilization in adulthood and childhood,

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which impairs the rehabilitation process, however, far fewer studies have been

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conducted analyzing atrophic response in infants. This work investigated first the

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morphological and molecular mechanisms involved in immobilization-induced

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atrophy in soleus muscles from rats at different stages of postnatal development [i.e.,

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weanling (WR) and adult (AR) rats] and, second, the role of autophagy in regulating

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muscle plasticity during immobilization. Hindlimb immobilization for 10 days

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reduced muscle mass and fiber cross-sectional area, with more pronounced atrophy in

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WR, and induced slow-to-fast fiber switching. These effects were accompanied by a

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decrease in markers of protein synthesis and an increase in autophagy. The Ub-ligase

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MuRF1 and the ubiquitinated proteins were upregulated by immobilization in AR

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while the autolysed form of μ-calpain was increased in WR. To further explore the

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role of autophagy in muscle abnormalities, AR were concomitantly immobilized and

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treated with colchicine, which blocks autophagosome-lysosome fusion. Colchicine-

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treated immobilized muscles had exacerbated atrophy and presented degenerative

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features. Despite Igf1/Akt signaling was downregulated in immobilized muscles from

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both age groups, Foxo1 and 4 phosphorylation was increased in WR. In the same

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group of animals, Foxo1 acetylation and Foxo1 and 4 content was increased and

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decreased, respectively. Our data show that muscle disorders induced by 10-day-

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immobilization occurs in both age-dependent and -independent manners, an

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understanding that may optimize treatment outcomes in infants. We also provide

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further evidences that the strong inhibition of autophagy may be ineffective for

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treating muscle atrophy.

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Keywords muscle atrophy, fiber type, Igf1/Akt/Foxo signaling, protein metabolism,

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autophagy.

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NEW & NOTEWORTHY STATEMENT

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Immobilization induces muscle maladaptations at different stages of postnatal

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development, but the cellular mechanisms involved in such effects are unclear. Our

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data show that the alterations in muscle proteostasis during immobilization occur in

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age-independent and -dependent manners and muscle disorders are aggravated by

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autophagy blockade with colchicine, inducing a myopathic profile. This

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understanding may help us optimize treatment outcomes in immobilized adults and

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infants and encourages the test of different autophagy inhibitors in muscle atrophy.

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INTRODUCTION

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Among several clinical settings, traumatic orthopedic injury is one of the leading

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causes of morbidity and mortality of both adults (32) and children (16). Skeletal

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muscle atrophy occurs frequently following injury and immobilization, which results

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in significant loss of force production and has a profound impact on rehabilitation

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process (2, 31, 42). Unfortunately, the treatment of muscle wasting under these

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conditions remains an unsolved problem, especially during childhood, in part, due to a

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lack of understanding of the cellular and molecular mechanisms responsible for the

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induction and maintenance of muscle loss.

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Muscle atrophy can affect specific fiber types and is frequently accompanied by a

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fiber type switching. For instance, muscle disuse, such as sciatic denervation or hind

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limb unloading, causes a more pronounced atrophy in slow, oxidative muscles

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enriched in type I fiber (e.g., soleus) with a slow-to-fast fiber type switching (36, 37).

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The mechanisms by which disuse induces muscle plasticity are uncertain and may

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involve a decrease in the rate of protein synthesis (55) and/or an increase in protein

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breakdown (7, 54). Skeletal muscle contains at least three systems for degrading

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proteins: Ca2+-dependent, ubiquitin (Ub)–proteasome (UPS), and autophagy-

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lysosome (ALS) proteolytic systems. Ca2+-dependent system includes two ubiquitous

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well-characterized cysteine proteases, μ-calpain and m-calpain, and their specific

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endogenous inhibitor, calpastatin (18). The contribution of this proteolytic system to

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muscle plasticity was evaluated in transgenic mice with muscle-specific

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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

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switch from a slow- to fast-twitch fiber type, which normally occurs in muscle

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unloading (53).

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Similar to the inhibition of Ca2+-dependent proteolysis, the in vivo blockade of

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proteasome by the treatment with VelcadeTM has partially prevented muscle atrophy

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by denervation and cast immobilization of the hind limb (1, 23). UPS usually

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degrades the majority of intracellular proteins and consists of ordered actions of

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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

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Ub onto proteins during muscle atrophy (5), which leads to the recognition and

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degradation of ubiquitinated proteins by proteasome. Unlike the UPS, ALS is

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primarily responsible for the degradation of most long-lived or aggregated proteins

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and cellular organelles. The main characteristic of ALS is the formation of

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autophagosome, which envelops substrates and delivers them to the lysosome for

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breakdown (46). Among the autophagy proteins, LC3 is critical for the elongation of

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isolation membranes and is localized to preautophagosomes and autophagosomes,

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making this protein a specific readout for autophagy (22). Despite the expression of

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several components and the activity of ALS have been found to be upregulated in

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muscle by a variety of atrophy situations (46, 59), muscle-specific deletion of the

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crucial autophagy gene Atg7 resulted in profound muscle wasting in basal condition

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and during denervation and fasting (29). Thus, the protective role of ALS inhibition in

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muscle atrophy conditions remains unclear.

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Interestingly, the activities of both UPS and ALS are upregulated by a common

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family of transcription factors, termed Forkhead box class O (Foxo) (28, 45). At basal

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state, the phosphorylation of Foxo by Akt, a central kinase that lies downstream of

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insulin-like growth factor-1/phosphatidylinositol-3-kinase (Igf1/PI3K) axis, induces

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attenuation of DNA binding and its translocation to the cytoplasm from the nucleus,

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leading to a decrease in the expression of Foxo-target genes (45, 51). Besides

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phosphorylation, Foxo proteins are also regulated through other post-translational

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modifications including acetylation (4, 49). By using genetic tools Bertaggia et al. (4)

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showed that Foxo3 is progressively acetylated, which induces its cytosolic

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localization and lower stability, during denervation and concomitantly Atrogin-1 is

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downregulated. In addition to inhibit proteolysis, Akt also promotes protein synthesis

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by recruiting several effectors such as mechanistic target of rapamycin (mTOR) and

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glycogen synthase kinase 3β (GSK3β) (47).

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The purpose of this study was to examine the morphological and molecular

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mechanisms involved in immobilization-induced atrophy in slow-twitch soleus

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muscles from female rats at different stages of postnatal development. Additionally,

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we aimed at investigating the role of autophagy in regulating muscle plasticity during

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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

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pharmacological blockade with colchicine (COL) markedly aggravates muscle

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abnormalities and induces a myopathic profile. Our data also provide evidences that

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the inhibition of autophagy induced by COL may be ineffective for treating muscle

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atrophy following immobilization.

130 131

METHODS

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Animals and experimental procedures

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Female weanling (21 days old, ~60g) and adult (81 days old, ~320g) Wistar rats were

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used in the experiments. The animals were housed in a room with a 12:12 h light-dark

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cycle and were given free access to water and a normal chow diet. For most

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experiments, the following four experimental groups were used: control weanling

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(CW), 10-day-immobilized weanling (IW), control adult (CA) and 10-day-

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immobilized adult rats (IA). The duration of immobilization was chosen because

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muscle atrophy occurred at this time point in both age groups (2, 12). The animals

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were submitted to immobilization (see Immobilization Model) for 10 days and then

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euthanized in a carbon dioxide chamber. Soleus muscles were removed, rolled in

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talcum powder, frozen in liquid nitrogen, and stored at -80ºC until further processing

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for the analysis of fiber cross-sectional area (FCSA) and immunofluorescence. For

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determination of mRNA (qPCR) and protein (western blot) levels of the markers of

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protein metabolism and signaling pathways, talcum powder was not used for freezing

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muscles. We chose to analyze soleus muscle, because previous studies (2, 7, 36)

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indicate that slow-twitch muscles like soleus undergo rapid and more pronounced

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atrophy than fast-twitch muscles in rodent models of muscle disuse. For the

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evaluation of autophagy flux in vivo, adult rats were treated or not with colchicine

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(COL; C9754; Sigma-Aldrich) at a dose of 0.4 mg/kg/day (ip) in the last three days of

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the immobilization (i.e., 8, 9 and 10 day). This protocol was originally proposed by Ju

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et al. (20) and modified by us by giving an additional injection of COL 1 h prior the

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euthanasia. Control groups received an equal volume of water (vehicle). In order to

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induce a chronic blockade of autophagy, weanling and adult rats were concomitantly

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submitted to immobilization and treatment with COL for 10 days. All experiments

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and protocols were performed in accordance with the ethical principles of animal

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research adopted by the Brazilian College of Animal Experimentation (COBEA) and

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were approved by Ribeirão Preto Medical School of the University of São Paulo –

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Ethical Commission of Ethics in Animal Research (CETEA; no 146/2012).

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Immobilization model

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Before the immobilization procedure, rats were anesthetized using an intraperitoneal

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injection of 4% chloral hydrate (4 ml/kg). The right hind limb was immobilized as

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previously described (2). Briefly, the upper part of the immobilization device is

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similar to a T-shirt made of viscolycra, which allows free movements of the head and

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forelimbs of the rat. The lower part of the device, divided into anterior and posterior

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sections, consisted of a stainless steel mesh with the margins wrapped with

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impermeable surgical tape. The anterior section was also wrapped with cotton lining

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to protect the anterior surfaces of the immobilized limb and hip. After that, the upper

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and lower portions of the device were joined with staples. This immobilization model

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allows daily adaptation according to the growth of the animals during immobilization

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and consists of maintaining the tibial-tarsal joint in maximum plantar flexion for a

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period of 10 consecutive days.

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Muscle preparation for histology

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Muscles were cut into 5-µm-thick transverse sections with a Leica CM1850 UV

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cryostat at -25°C (Leica Microsystems, Wetzlar, Germany); tissue sections were then

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placed on 26×76 mm slides and were used for hematoxylin and eosin (H&E) and

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Picrosirius stainings, myofibrillar adenosine tri-phosphatase (mATPase) reaction and

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immunohistochemistry.

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Histological and morphometric analysis

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The tissue sections were stained with hematoxylin and eosin (H&E) and the images

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were captured using an optical microscope (Leica DM 2500). Fiber cross-sectional

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area (FCSA, expressed in µm2) was calculated by counting five random fields (X400

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magnification) of each muscle using ImageJ software (version 1.45s, National

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Institutes of Health, USA).

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The analysis of the connective tissue area was performed in slides stained by

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Picrosirius. The images were captured by a high-resolution camera (Leica DFC290,

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Leica Microsystems, Frankfurt, Germany) connected to an optical microscope (Leica

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DMR 2500) coupled to a polarizing filter showing the birefringence of collagen fibers

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(X200 magnification). The quantification of connective tissue was determined by

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counting mean area of three random fields of each muscle using the Leica QWin V3

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software.

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As shown in Figures 1K-N, four fiber types (I, IIA, IIC and IIX) were determined by

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mATPase (E.C.2.1.3.5.7.9.1) histochemistry reaction (19) in acid and alkaline

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medium (pH 4.35, 4.65 and 9.8). For the analysis of fiber type distribution and FCSA,

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the number of each fiber type and myofiber size were determined by counting three

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random fields of each muscle (X200 and X400 magnification, respectively, for adult

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and weanling rats) using LAS software (Leica Microsystem) as described previously

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(11). The images were captured by Leica DM 2500 light microscope.

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Immunohistochemistry and fluorescence microscopy

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A single immunofluorescence analysis of myosin heavy chain (MHC) isoforms was

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performed with primary antibodies against MHCI (1:40) and MHCIIA (1:5). The

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secondary antibodies used were goat anti-mouse-Red Alexa Fluor 568 (against MHC

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I; 1:250) and goat anti-mouse IgG - Blue Alexa Fluor 350 (against MHC IIA; 1:250).

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Muscle slices were fixed in Xpress molecular fixative (Sakura Finetek, Alphen aan

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den Rijn, Netherlands), washed with phosphate-buffered saline (PBS) and then

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blocked with 10% normal goat serum (Vector Laboratories, Road Burlingame,

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California, USA) in PBS. The slices were blocked with Avidin (Avidin/Biotin

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Blocking Kit, Vector Laboratories, Road Burlingame, California, USA), washed with

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PBS, and incubated with the primary antibody for 2h at 37°C. After washing with

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PBS, the slices were incubated with a secondary antibody, washed with PBS and

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mounted with Prolong Gold Antifade without DAPI (Invitrogen). For the analysis of

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FCSA, myofiber size was determined by counting three random fields (X200

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magnification) of each muscle using ImageJ software.

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Western blotting analysis

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Soleus muscles were homogenized in 50 mM Tris–HCl buffer (pH 7.4) containing

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150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% sodium deoxycholate, 1% SDS,

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10 mM sodium pyrophosphate, 100 mM sodium fluoride, 10 mM sodium

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orthovanadate, 5 µg/ml of aprotinin, 1 mg/ml of leupeptin, and 1 mM phenylmethyl-

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sulfonyl fluoride (PMSF) at 4°C. The homogenate was centrifuged at 21,000xg at 4°C

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for 20 min, and the supernatant was retained. Protein content was determined using

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BSA as a standard (26). An equal volume of sample buffer (20% glycerol, 125 mM

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Tris–HCl, 4% SDS, 100 mM dithiothreitol, 0.02% bromophenol blue, pH 6.8) was

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added to the supernatant, and the mixture was boiled. Thirty to fifty micrograms of

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total protein was separated by SDS-PAGE, transferred to nitrocellulose membranes,

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and blotted with anti-LC3-I/II (1:1,000), anti-p62 (1:1,000), anti-atrogin-1 (1:750),

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anti-MuRF-1 (1:750), anti-calpastatin (1:1,000), anti-m-calpain (1:750), anti-µ-

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calpain (1:750), anti-S6 (1:1,000), anti-eIF4E (1:1,000), anti-4EBP1(1:1,000), anti-

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GSK3β (1:1,000), anti-Ubiquitin (1:1,500), anti-20S proteasome β1 subunit (1:750),

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anti-20S proteasome β3 subunit (1:1,000), anti-20S proteasome β5 subunit (1:750),

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anti-Akt (1:750), anti-Foxo3a (1:750), anti-Foxo1 (1:1,000), anti-Foxo4 (1:1,000),

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anti-phospho (p)-[Ser473]-Akt (1:500), anti-p-[Thr308]-Akt (1:500), anti-p-[Ser9/21]-

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GSK3β/α (1:1,000), anti-p-[Ser235/236]-S6 (1:1,000), anti-p-[Thr70]-4EBP1(1:1,000),

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anti-p-[Ser209]-eIF4E (1:1,000), anti-p-[Ser256]Foxo1 (1:500), anti-p-[Ser193]Foxo4

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(1:500),

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[Lys259/262/271]Foxo1 (1:250) and β-actin (1:1,000). Primary antibodies (Ab) were

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detected using peroxidase-conjugated secondary Ab (1:1,000 for anti-p-[Ser473]-Akt,

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anti-p-[Thr308]-Akt, anti-Akt, anti-p-[Ser193]Foxo4, anti-ac-[Lys259/262/271]Foxo1, anti-

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p-[Thr24]Foxo1/[Thr32]Foxo3a, anti-Foxo3a, anti-Foxo4, anti-Foxo1, anti-S6, anti-

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eIF4E, anti-4EBP1, anti-GSK3β,

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anti-m-calpain,

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[Ser235/236]-S6, anti-p-[Thr70]-4EBP1, anti-p-[Ser209]-eIF4E, anti-atrogin-1, anti-

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MuRF-1, 1:5,000 for β-actin and 1:10,000 for anti-Ubiquitin, anti-20S proteasome β1

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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-

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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

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Soleus muscles were harvested and immediately frozen in liquid nitrogen. RNA was

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subsequently isolated from individual skeletal muscles using Trizol (Invitrogen).

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Reverse transcription of RNA to cDNA was performed using 1 µg of total cellular

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RNA, 20 pmol of oligo(dT) primer (Invitrogen), and Advantage ImProm-II reverse

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transcriptase (Promega, Madison, WI). Quantitative (real-time) PCR was performed

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using an ABI7000 sequence detection system (Applied Biosystems, Foster City, CA),

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a SuperScript III Platinum SYBR Green One-Step RT-qPCR Kit with ROX

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(Invitrogen), and primers for rat Igf1 (forward 5’-GCT TGC TCA CCT TTA CCA

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GC-3’ and reverse 5’-AAT GTA CTT CCT TCT GGG TCT-3’) and Rpl39 (forward

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5’-CAA AAT CGC CCT ATT CCT CA and reverse 5’-AGA CCC AGC TTC GTT

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CTC CT). The relative quantification of mRNA levels was plotted as the fold-increase

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in comparison to the values of the respective control group. Igf1 transcript was

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normalized to Rpl39 levels, and the mRNA levels were calculated using the standard

266

curve method (9).

267 268

Antibodies, drugs and reagents

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Rabbit

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calpastatin, anti-m-calpain, anti-µ-calpain, anti-LC3-I/II, anti-p-[Ser9/21]-GSK3β/α

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(1:1,000), anti-p-[Ser240/244]-S6, anti-p-[Thr70]-4EBP1 and anti-p-[Ser209]-eIF4E were

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purchased from Cell Signaling Technology (Danvers, MA). Rabbit anti-atrogin-1 and

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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

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from Merck Millipore (Darmstadt, Germany) and MHCIIA (#SC-71, 2F7) were from

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the Developmental Studies Hybridoma Bank (University of Iowa, Iowa). The

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secondary Abs for immunofluorescence were from Invitrogen (Life Technologies,

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Carlsbad, CA). All other drugs and reagents were purchased from Sigma-Aldrich (St.

279

Louis, MO), Thermo Scientific HyClone (Pittsburgh, PA), Invitrogen (Carlsbad, CA),

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Calbiochem EMD Biosciences (La Jolla, CA), or Amersham Biosciences

281

(Piscataway, NJ).

282 283

Image editing

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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

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Extended (Adobe) and Microsoft PowerPoint (Microsoft).

287 288

Statistical analysis

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The data are presented as the mean ± standard error (SE). The means from different

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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-

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way ANOVA followed by a Holm-Sidak post hoc test. P ≤ 0.05 was taken as the

293

criterion for significance.

294 295

RESULTS

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Immobilization induces skeletal muscle atrophy and fiber type transition in weanling

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and adult rats

13 298

Figure 1 displays the wet mass, the ratio of muscle mass-to-body mass, and the fiber

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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

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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

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(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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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