**2. Autophagy, an intracellular system that delivers cytoplasmic components to lysosome for degradation**

#### **2.1. The autophagy pathways**

**1. Introduction**

172 Muscle Cell and Tissue

in fiber number [1].

Skeletal muscle exhibits remarkable adaptive capabilities in response to various stimuli such as loading conditions (resistance training, microgravity), contractile activity (electrical stimulations, endurance exercise), environmental factors (altitude exposure), or nutritional interventions. To access this great capacity, a plethora of quantitative and functional adaptations are involved. Changes in the size of adult muscle, in response to these external stimuli, are mainly due to the growth of individual muscle fibers rather than an increase

The control of muscle mass is dependent upon a balance between anabolic and catabolic processes. Hypertrophy is associated with increased protein synthesis, while atrophy is characterized by increased degradation of muscle proteins and/or a decrease in protein translation. The initiation of protein synthesis is mainly mediated by a signaling pathway in which the mammalian/mechanistic target of rapamycin complex 1 (MTORC1), a multiprotein complex composed of MTOR (mammalian/mechanistic target of rapamycin), RPTOR (regu‐ latory-associated protein of MTOR), mLST8/GβL (MTOR-associated protein LST8 homolog), DEPTOR (DEP domain containing MTOR-interacting protein), and PRAS40 (proline-rich Akt substrate of 40 kDa) [2,3]. MTORC1 by phosphorylating its substrates S6K1 (ribosomal protein p70S6 kinase 1) and 4E-BP1 (eukaryotic translation initiation factor 4E-binding protein 1) controls skeletal muscle protein translation and hypertrophy [4-7]. The Insulin signaling pathway leads to the activation of MTORC1 through the activation of the kinases PI3K (phosphatidylinositol 3-kinase), PDK1 (phosphoinositide-dependent kinase-1), and Akt. Akt, also known as protein kinase B (PKB), inactivates tuberous sclerosis complex 1/2 (TSC1/2), promoting MTOR activation by Rheb-GTP [8-10]. Akt also phosphorylates and inactivates the glycogen synthase kinase 3β (GSK3β), resulting in the activation of the eukaryotic translation

Muscle atrophy leads to a state of weakness and emaciation of the body, which is encountered, for example, in the terminal phase of certain diseases or chronic infections such as cancer, AIDS, diabetes, bacterial infections, and nerve degeneration [12]. Muscle atrophy is also observed during aging, immobilization, and stress or trauma to the muscle and is associated with increased proteolysis. Protein degradation is essentially modulated by two conserved path‐ ways: the ATP-dependent ubiquitin-proteasome system and the autophagy pathways.

The first one has been particularly involved in the degradation process after the discovery of two E3 ubiquitin ligases (E3 ligases), MAFbx/atrogin-1 (muscle atrophy F-box) and MuRF1 (muscle RING finger-1), which are both overexpressed in various models of atrophy (fasting, cancer, diabetes, immobilization, and other stresses) [13,14]. The invalidation of these proteins confers a resistance to certain types of induced atrophy, suggesting a critical role in the catabolism for the ubiquitin-proteasome pathway [13]. The function of E3 ligases is to ubiq‐ uitinate specific proteins to target them for recognition by the 26S proteasome where they are eliminated. Other E3 ligases like zinc-finger protein 216 (ZNF216), the mitochondrial E3 ubiquitin protein ligase 1 (Mul1), and the tripartite motif-containing protein 32 (Trim32) have

initiation factor 2B (eIF2B) and increased protein synthesis [5,11].

been shown to play an important role in skeletal muscle atrophy [15-17].

#### *2.1.1. Description of the system*

Autophagy has been discovered during nutrient privation and can be referred to as "selfeating" as cells degrade their own constituents to maintain cellular homeostasis in response to various injuries like starvation and hypoxia and in pathological conditions, including cancer, muscular dystrophy, and neurodegenerative diseases. One purpose of the starvation-in‐ duced autophagy is to degrade materials to provide amino acids and free fatty acids in order topreservemetabolismandATPlevelswhenextracellularnutrients reachhazardous lowlevels [22]. Moreover, autophagy also eliminates protein aggregates as well as unwanted and dysfunctional organelles. The term autophagy embraces macroautophagy, microautophagy, and chaperone-mediated autophagy that we will describe hereafter.

Macroautophagyandmicroautophagyareconservedfromyeasttohumans,andtheseprocesses were originally described as bulk degradation mechanisms. However, these two processes can be selective for targeting different organelles, and we distinguish mitophagy selective for degradation of mitochondria; pexophagy, selective for degradation of peroxisomes; xenopha‐ gy, selective for degradation of intracellular bacteria and virus; reticulophagy, selective for endoplasmic reticulum; heterophagy, selective for substances taken in by phagocytosis; golgiphagy, selective for Golgi apparatus; ribophagy, selective for ribosomes; crinophagy, specific for the contents (proteins, peptides) of secretory granules; glycophagy, selective for glycogen; and lipophagy, selective forlipid droplets [23-29]. Among these different varieties of autophagy, mitophagy has been the most studied in the last decade, and this process involves notably two Parkinson's disease factors, the RING-between-RING E3 ligase Parkin and the mitochondrialkinasePINK1 (PTEN-inducedputativekinaseprotein1),PTENbeing"phospha‐ tase and tensin homolog" [30,31]. After mitochondrial potential depolarization, PINK1 promotes Parkin activation through phosphorylation of its ubiquitin-like domain [32]. In addition to these factors, the mitochondrial E3 ligase Mul1 can also be involved in mitophagy by the degradation of the mitochondrial fusion protein Mfn2 (mitofusin-2) and the stabiliza‐ tion of the dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation [33].

Autophagy by providing a turnover of the cellular components plays a central role in the homeostasis ofthe cell.It is a key mechanism by which a starving cellreallocates nutrients from unnecessary to more-essential processes. Macroautophagy, microautophagy, and chaperonemediated autophagy lead cytoplasmic substrates inside lysosomes in which their contents are digested by a battery of acidic hydrolases [34]. Four essential ubiquitous proteases have been identified: the cathepsins B, D, H, and L [35,36]. High levels of these cathepsins are expressed in tissues exhibiting high rates of protein turnover like the kidney, spleen, liver, or placenta, while low concentrations of cathepsins are found in tissues with lower protein turnover as skeletal muscles [37-40]. Similar enzymatic properties were reported for different muscles, independent of their metabolic and contractile type. However, their concentrations differ according to the fiber type. Indeed, slow-twitch oxidative muscles exhibit higher levels of cathepsins than the fast-twitch glycolytic muscles [35], suggesting a more important activity of this system in oxidative muscle. This data is in agreement with the fact that oxidative muscles present a more important protein turnover and a greater translational activity than glycolytic muscles.

Althoughskeletalmuscle expresses cathepsinsB,D,H,andL,theyplaydistinctive roles.During fusion of myoblasts into myotubes, several groups have reported an increase in the expres‐ sion and activity of lysosomal cathepsins, in particular cathepsin B [41-46]. Several studies showed that the expression of cathepsin L is induced during various forms of skeletal muscle atrophy including starvation [12,36,47-49]. Increase in cathepsin D activity has been reported in muscles of dystrophic mice and chicken and in muscles of patients with Duchenne muscu‐ lar dystrophy [35,50].

#### *2.1.2. Microautophagy*

Microautophagy is localized directly at the level of the lysosome which directly engulfs cytosol components by invagination, protrusion, and/or elimination of the lysosomal limiting mem‐ brane. It is implicated in the degradation of long half-life proteins in numerous cell types and does not respond to classical stimuli inducing chaperone-mediated autophagy and macroau‐ tophagy [51].Incontrasttomacroautophagy,microautophagyhasnot beenextensivelystudied in skeletal muscle, and its functions in muscle proteolysis have to be more characterized [35].

### *2.1.3. Chaperone-mediated autophagy*

Chaperone-mediated autophagy (CMA) is a selective form of autophagy that has only been described in mammalian cells to date [52,53]. In this form of autophagy, only cytosolic proteins that possess the consensus pentapeptide Lys-Phe-Glu-Arg-Gln (KFERQ) are recognized. The

KFERQ-like sequence is recognized by the heat-shock cognate protein of 73 kDa (hsc73) then targeted to lysosomes for degradation. This targeting needs the binding of the complex protein substrates hsc73 to the lysosome-associated membrane protein type 2A (LAMP-2A) and to a multi-molecular chaperone complex including hsp40, hsp70, and hsp90 at the cytosolic side of the lysosomal membrane [54]. LAMP-2A is a glycoprotein present at the lysosomal mem‐ brane which acts as a CMA receptor. The substrates are then unfolded and translocated across the lysosomal membrane with the hsc73protein.Unlike otherforms of autophagy, CMAis very selective in substrate degradation and cannot eliminate organelles [55-57].

The use of an antibody directed to the KFERQ amino acid sequence substrates showed that proteins containing this sequence are conserved in skeletal muscle during starvation, while they are degraded in the liver and heart [58]. Moreover, the absence of consensus sequence in most myofibrillar proteins indicates that this degradation pathway is not implicated in their degradation. However, Nishino and colleagues showed that LAMP-2 deficiency is the primary defect in human Danon disease, a pathology characterized by myopathy and cardiomyopathy with massive accumulation of autophagic vacuoles [59,60].Thus, as microautophagy, CMA has to be more characterized in the context of muscle atrophy.

#### *2.1.4. Macroautophagy*

autophagy, mitophagy has been the most studied in the last decade, and this process involves notably two Parkinson's disease factors, the RING-between-RING E3 ligase Parkin and the mitochondrialkinasePINK1 (PTEN-inducedputativekinaseprotein1),PTENbeing"phospha‐ tase and tensin homolog" [30,31]. After mitochondrial potential depolarization, PINK1 promotes Parkin activation through phosphorylation of its ubiquitin-like domain [32]. In addition to these factors, the mitochondrial E3 ligase Mul1 can also be involved in mitophagy by the degradation of the mitochondrial fusion protein Mfn2 (mitofusin-2) and the stabiliza‐ tion of the dynamin-related protein 1 (DRP1), resulting in mitochondrial fragmentation [33].

Autophagy by providing a turnover of the cellular components plays a central role in the homeostasis ofthe cell.It is a key mechanism by which a starving cellreallocates nutrients from unnecessary to more-essential processes. Macroautophagy, microautophagy, and chaperonemediated autophagy lead cytoplasmic substrates inside lysosomes in which their contents are digested by a battery of acidic hydrolases [34]. Four essential ubiquitous proteases have been identified: the cathepsins B, D, H, and L [35,36]. High levels of these cathepsins are expressed in tissues exhibiting high rates of protein turnover like the kidney, spleen, liver, or placenta, while low concentrations of cathepsins are found in tissues with lower protein turnover as skeletal muscles [37-40]. Similar enzymatic properties were reported for different muscles, independent of their metabolic and contractile type. However, their concentrations differ according to the fiber type. Indeed, slow-twitch oxidative muscles exhibit higher levels of cathepsins than the fast-twitch glycolytic muscles [35], suggesting a more important activity of this system in oxidative muscle. This data is in agreement with the fact that oxidative muscles present a more important protein turnover and a greater translational activity than glycolytic

Althoughskeletalmuscle expresses cathepsinsB,D,H,andL,theyplaydistinctive roles.During fusion of myoblasts into myotubes, several groups have reported an increase in the expres‐ sion and activity of lysosomal cathepsins, in particular cathepsin B [41-46]. Several studies showed that the expression of cathepsin L is induced during various forms of skeletal muscle atrophy including starvation [12,36,47-49]. Increase in cathepsin D activity has been reported in muscles of dystrophic mice and chicken and in muscles of patients with Duchenne muscu‐

Microautophagy is localized directly at the level of the lysosome which directly engulfs cytosol components by invagination, protrusion, and/or elimination of the lysosomal limiting mem‐ brane. It is implicated in the degradation of long half-life proteins in numerous cell types and does not respond to classical stimuli inducing chaperone-mediated autophagy and macroau‐ tophagy[51].Incontrasttomacroautophagy,microautophagyhasnot beenextensivelystudied in skeletal muscle, and its functions in muscle proteolysis have to be more characterized [35].

Chaperone-mediated autophagy (CMA) is a selective form of autophagy that has only been described in mammalian cells to date [52,53]. In this form of autophagy, only cytosolic proteins that possess the consensus pentapeptide Lys-Phe-Glu-Arg-Gln (KFERQ) are recognized. The

muscles.

174 Muscle Cell and Tissue

lar dystrophy [35,50].

*2.1.2. Microautophagy*

*2.1.3. Chaperone-mediated autophagy*

Macroautophagy, often referred to as autophagy or autophagosome-lysosome system, is an evolutionarily conserved intracellular system that coordinates and oversees the degradation of damaged organelles as mitochondria, peroxisomes, or ribosomes, intracellular pathogens, and unused long-lived proteins [61]. More than 30 autophagy-specific genes (Atgs) have been identified and are known to facilitate the sequestration of cytoplasmic substrates into a doublemembrane vesicle called autophagosome or autophagic vacuole. Atgs are essential mediators of autophagy, by controlling the formation of the autophagosome. Autophagosome fuses with lysosome to form an autolysosome (also called autophagolysosome).

### **2.2. Macroautophagy: Autophagosome formation**

### *2.2.1. Initiation*

Macroautophagy (autophagy) is initiated in response to a multitude of factors including nutrient deprivation and oxidative stress. The activation of autophagy during muscle wasting was shown by the accumulation of autophagosomes in muscles of fasted transgenic GFP-LC3 mice [62]. Studies showed that during starvation-induced atrophy, FoxO3a (forkhead box class O3a) regulates the transcription of several Atgs, including Atg4B, LC3B (microtubuleassociated protein 1A/1B-light chain 3B), Beclin-1, Vps34 (vacuolar protein sorting 34)/PI3K class III, Gabarapl1 (GABAA receptor-associated protein-like 1), Atg12, and Ulk2 (unc-51-like kinase 2) [63,64]. Initiation of the autophagy processes involves the activation of the unc-51 like kinase 1 (Ulk1, also called Atg1 in yeast)/Atg13/FIP200/Atg101 and the Beclin-1/Vps34 complexes. These proteins operate in conjunction with several Atgs to mediate the assembly of the autophagosomal membrane [65-68]. Ulk1 also phosphorylates Beclin-1 at Ser-14 following amino acid withdrawal, and this stage is necessary for the Vps34 lipid kinase activation and full autophagy induction [69]. In yeast and mammalian cells, Atg1 or Ulk1 (respectively) activity is suppressed under nutrient-rich conditions by MTORC1 (phosphory‐ lation of Ulk1 at Ser-757) [65,70-73]. In addition, MTOR inhibition and its subsequent dissoci‐ ation from Ulk1 are critical for Beclin-1/Vps34 complex activation by Ulk1 [69].

#### *2.2.2. Maturation of autophagosomes*

The maturation and completion of the autophagic vacuole is facilitated by a ubiquitin-like conjugation-signaling cascade that culminates with the binding of phosphatidylethanolamine (PE) to the cytosolic form of LC3 (LC3-I) to form LC3-II. LC3, a mammalian homolog of yeast Atg8, is a protein with a molecular mass of 17 kDa that is distributed ubiquitously in mam‐ malian tissues. Two other LC3 homologs are GABAA receptor-associated protein (Gabarap) and Golgi-associated ATPase enhancer (GATE-16). LC3-phosphatidylethanolamine conjugate (LC3-II) is recruited to the autophagosomes [74,75]. LC3-II plays here a structural role that allows the elongation and the formation of the mature autophagosome. The mature autopha‐ gosome fuses with the lysosome to form an autolysosome (Fig.1). Concomitantly, LC3-II in autolysosomal lumen is degraded by lysosomal hydrolases; thus, turnover of the autophago‐ somal protein LC3-II reflects autophagic activity [76]. LC3 also interacts with the autophagy adapter p62/SQSTM1 (sequestosome 1), which contains multiple apparent protein-protein interaction domains. p62 binding to LC3 permits the entry of ubiquitinated cargo into the autophagosome [77]. In addition, the Atg12-Atg5-Atg16 complex plays an important role in the maximal promotion of LC3 lipidation and autophagy induction [78].

**Figure 1.** Processing of the macroautophagy system

#### **2.3. Implication of autophagy in cell homeostasis and disease**

activation and full autophagy induction [69]. In yeast and mammalian cells, Atg1 or Ulk1 (respectively) activity is suppressed under nutrient-rich conditions by MTORC1 (phosphory‐ lation of Ulk1 at Ser-757) [65,70-73]. In addition, MTOR inhibition and its subsequent dissoci‐

The maturation and completion of the autophagic vacuole is facilitated by a ubiquitin-like conjugation-signaling cascade that culminates with the binding of phosphatidylethanolamine (PE) to the cytosolic form of LC3 (LC3-I) to form LC3-II. LC3, a mammalian homolog of yeast Atg8, is a protein with a molecular mass of 17 kDa that is distributed ubiquitously in mam‐ malian tissues. Two other LC3 homologs are GABAA receptor-associated protein (Gabarap) and Golgi-associated ATPase enhancer (GATE-16). LC3-phosphatidylethanolamine conjugate (LC3-II) is recruited to the autophagosomes [74,75]. LC3-II plays here a structural role that allows the elongation and the formation of the mature autophagosome. The mature autopha‐ gosome fuses with the lysosome to form an autolysosome (Fig.1). Concomitantly, LC3-II in autolysosomal lumen is degraded by lysosomal hydrolases; thus, turnover of the autophago‐ somal protein LC3-II reflects autophagic activity [76]. LC3 also interacts with the autophagy adapter p62/SQSTM1 (sequestosome 1), which contains multiple apparent protein-protein interaction domains. p62 binding to LC3 permits the entry of ubiquitinated cargo into the autophagosome [77]. In addition, the Atg12-Atg5-Atg16 complex plays an important role in

ation from Ulk1 are critical for Beclin-1/Vps34 complex activation by Ulk1 [69].

the maximal promotion of LC3 lipidation and autophagy induction [78].

*2.2.2. Maturation of autophagosomes*

176 Muscle Cell and Tissue

**Figure 1.** Processing of the macroautophagy system

Autophagy plays a prominent role in the maintaining of cell homeostasis by selectively eliminating protein aggregates, damaged organelles, and other nonactive cellular debris [79,80]. This process is required for normal cellular function, but it is increasingly apparent that it can have both beneficial and detrimental effects on cells and tissues, depending on the origin of its activation [81]. Physiological function of basal autophagy in maintaining tissue homeostasis has been demonstrated in several tissues as the brain, liver, heart, striated muscle, intestine, pancreas, and adipose tissue [82-89]. Furthermore, exciting reports suggest that autophagy may contribute to counteract the deleterious effects of aging by limiting the deposition of aggregated proteins and damaged mitochondria [90-92]. By blocking apoptosis, autophagy preserves cell survival by providing endogenous metabolites when exogenous substrates are lacking [93]. Thus, at regular levels of activation, autophagy may represent the first step to restore homeostasis. However, when the autophagic capacity is submerged, apoptosis takes over [94]. Nevertheless, the relationship between autophagy and apoptosis appears to be extremely complex, and additional data are necessary to clarify the situation, especially in the context of disease.

In numerous pathologies as diabetes, obesity, cancer, heart failure, and neurodegenerative, infectious, and inflammatory disease, autophagy activity is affected [34,95,96]. For instance, it has been reported that the lack in Beclin-1 expression decreases autophagy flux and leads to increased risk of breast and prostate cancers [97]. However, the systematic beneficial effect of autophagy should be tempered. Thus, the role of autophagy in cancer is ambivalent, and this process can be involved in both the promotion and the prevention of this disease. In the first stage of the malignancy (i.e., tumor initiation), inhibition of autophagy may allow the growth of initial cancerous cells, and thus autophagy can act as a suppressor of cancer [98,99]. When cancer is established, transformed cells may need autophagy to survive, especially in nutrientlimiting condition [100]. In addition, for patient undergoing treatment such as chemotherapy, cancer cells could use autophagy to protect themselves from the stress induced by the therapy. Other reports indicate also that glycogen storage disease type II (also called Pompe disease) – an autosomal recessive metabolic disorder – is a pathology attributable in part to mutations of Atgs. In this disease, muscle and nerve cells are damaged by an accumulation of glycogen into the lysosomes caused by a deficiency of the lysosomal acid alpha-glucosidase enzyme [101,102]. In many neurodegenerative disorders such as Parkinson's, Huntington's, and Alzheimer's diseases, accumulation of autophagic vesicles has been also observed [103-105]. Regarding Alzheimer's disease, it has been reported that Atg7 influences the accumulation of amyloid β (Aβ) peptides, resulting in aggregation into plaques in the brain [106]. In this model, autophagy seems to participate to the disease progression since it is involved in the generation of Aβ peptides.

In summary, by removing misfolded proteins and abnormal organelles, autophagy can be considered as a critical mechanism for cell protection. On the contrary, by destroying excessive fraction of cytosol and organelles, too high levels of autophagy represent a side mechanism responsible of the initiation of pathologies [94,107].

#### **2.4. The roles of autophagy in skeletal muscle**

Compared to other tissues like the liver or pancreas, autophagy in skeletal muscle exhibits a low protein turnover and a small size of autophagosomes. These peculiar characteristics have probably constituted a brake for the detection of autophagy in this tissue for a long time. Associated to the use of transgenic mice expressing LC3 fused with GFP, autophagy process can be now easily visualized [62]. Conversely to liver or pancreas in which autophagy is activated transiently for a few hours, in skeletal muscle, autophagy can be activated for several days [62]. As it was shown in other tissues, muscular autophagy is activated by nutrient deprivation or by the absence of growth factors [108].

Although it was reported that the mRNAs coding for Atgs are present in abundance in skeletal muscle [109], the role and the regulation of basal autophagy have been poorly characterized in this tissue until recently. In order to assess the function of autophagy in skeletal muscle, Masiero and colleagues performed experiments on mice deprived of the Atg7 gene, a gene necessary to the unfolding of the autophagy program [86,110]. Importantly, mice showed obvious signs of muscular weakness and atrophy exacerbated during ageing. Mice presented an accumulation of degraded proteins and free radicals, a deterioration of the internal cellular structures, and an activation of the apoptotic program. The authors clearly defined that inhibition of basal autophagy does not protect from skeletal muscle atrophy induced by denervation or starvation, but on the contrary, contributes to its degeneration. Similar muscle alterations have been obtained in muscle-specific Atg5-/- mice [87], confirming the necessity to have regular autophagic flux in the cells, even during atrophy.

In many conditions varying from fasting, denervation, inactivity, microgravity, various pathologies as cancer, AIDS, sepsis, diabetes, cardiac failure, and myopathies, autophagy is overactive and pathologic, thus leading altered metabolism and muscle loss [81,108,111]. Contribution of autophagy to muscle loss begins to be clarified with the use of different animal models and innovative techniques. Inactivation of autophagic flux by LC3 silencing partially prevents FoxO3-mediated muscle atrophy [63] and atrophy caused by the expression of mutant SOD1 G93A in skeletal muscle [112]. In another model, atrophy induced by L-type calcium channel (DHPR) inactivation is linked to the expression of autophagic genes including LC3, Vps34, BNIP3 (BCL2/adenovirus E1B 19 kDa protein-interacting protein 3), and cathepsin L (for the lysosome) [113]. During sepsis, an upregulation of autophagy was found in parallel to mitochondrial injury and decreased biogenesis, especially in locomotor muscles [114]. Reactive oxygen species overproduction by altered mitochondria is now considered as a critical signal for the promotion of skeletal muscle autophagy, thus finding an opening to practical prospects for treatment of disease [115]. Accordingly, antioxidant supplement can lead to an inhibition of skeletal muscle autophagy through a reduction of oxidative stress and an increase in antioxidant capacity [116]. Taken together, these recent results confirm that excessive activation of autophagy is a critical factor for muscle wasting determinism, and strategies attempted to control autophagy level will be promising.

Data concerning sarcopenia are also specific. Sarcopenia is an age-related loss of muscle mass and strength, which is associated with increased autophagy, apoptosis, and exacerbated proteolysis [117]. Elevated peroxisome proliferator-activated receptor-coactivator α (PGC-1α) expression in muscle during aging contributes to reduce the proteolytic activity associated with atrophy. In sarcopenia, attenuation of the degradative processes and the maintenance of mitochondrial function contribute to the preservation of muscle integrity [118].

In summary, as already described for cancer cells, autophagy in muscle is a complex process that can be, according to its activation level, beneficial or deleterious. During disease, the systematic question is to determine whether autophagy plays a protective function, has a causative role, or is a result of the disease process itself.
