EFFECTS OF ANTIOXIDANT SUPPLEMENTATION ON HYPOACTIVITY-INDUCED

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Intermuscular adipose tissue accumulation

The loss of strength associated with muscle deconditioning is higher than the rate of atrophy, indicating that other factors are involved in muscle weakness. Indeed, a longitudinal 5-years study in aged people, showed a decrease of muscle strength 2 to 5 fold higher than the loss of muscle mass (Delmonico et al., 2009; Seene & Kaasik, 2012). In the context of real and simulated microgravity, the review of Di Prampero & Narici (2003) also described losses of muscle strength largely superior to those of muscle mass. It emphasizes the importance of other factors such as the accumulation of fat infiltration in strength’s loss (O Addison, Marcus, Lastayo, & Ryan, 2014; Brioche et al., 2016). Commonly called IMAT (InterMuscular Adipose Tissue), these infiltrations are composed by adipocytes localized under muscle epimysium, between fascicles and/or muscle fibers, and represent adipocytes clusters localized outside muscular cells (Vettor et al., 2009). IMAT should not be confounded with lipid droplets, the energy stocks situated within muscular cells.
IMAT is naturally present in humans. However, an increase of its content is harmful for the muscle well-functioning. Indeed, IMAT accumulation has been observed in various situations, all tightly linked to muscle deconditioning, such as: type II diabetes (Goodpaster et al., 2003; Goodpaster, Thaete, & Kelley, 2000b), aging (Brioche et al., 2016; Ryall, Schertzer, & Lynch, 2008; M. Y. Song et al., 2004), denervation or tendinous rupture (Dulor et al., 1998; H. M. Kim, Galatz, Lim, Havlioglu, & Thomopoulos, 2012). Nonetheless, it has been demonstrated that IMAT accumulation also occurred in young and healthy individuals after a reduction in physical activity of Manini et al. (2007) highlighted in healthy subjects an increase of 15% and 20% in thigh and calf IMAT after 4 weeks of hypoactivity induced by unilateral lower limb suspension model. Moreover, the review of Addison et al. (2014) underlined a correlation between activity levels and IMAT quantity in healthy subjects. By this way, the more sedentary someone is, the more likely is to have a higher IMAT content. An increase in IMAT development has negative repercussions on metabolism, muscular power, and mobility of the subjects. Literature also demonstrated strong correlations between IMAT levels and insulin-resistance (Goodpaster et al., 2000b). Globally, muscular function alterations induced by fat cells infiltration negatively impacts on physical performance and locomotion (O Addison et al., 2014; Goodpaster et al., 2008). The study of Tuttle et al. (2012) illustrated this phenomenon showing an inverse relation between IMAT content in calf and a speed reached in a 6 minutes walking test.
The origin and role of IMAT are not completely understood. First works in animals characterized adipogenic progenitors called FAPs (fibro-adipogenic progenitors) in the interstitial muscle space. These mesenchymal stem cells, expressing the receptor PDGFRα, are able to differentiate in fibrous or adipose tissue (Judson, Zhang, & Rossi, 2013; Penton, Thomas-Ahner, Johnson, McAllister, & Montanaro, 2013; Uezumi et al., 2011). In vitro and in vivo studies in mice demonstrated the primordial role of PDGFRα+ cells in the development and the accumulation of IMAT within muscle tissue (Boppart, De Lisio, Zou, & Huntsman, 2013; Uezumi, Fukada, Yamamoto, Takeda, & Tsuchida, 2010). The study of Uezumi et al. (2014) conducted in human allowed the identification and characterization of mesenchymal progenitors expressing PDGFRα in muscular tissue. In the case of muscle deconditioning, mechanisms leading to the possible engagement of FAPs in an adipogenic linage remain unknown. Nonetheless, it seems that modifications surrounded the vascular bed could be responsible for the engagement of various progenitors in an adipogenic linage. Subsequently, a cascade of signalization involving various transcription factors (STAT5, SREBP-1c, KLFs, PPARγ, C/EBPs) allow the growth of pre-adipocytes into mature adipose cells, to finally lead to IMAT accumulation (U. A. White & Stephens, 2010).

Dysregulation of protein balance: molecular pathways implicated

Molecular signaling involved in the regulation of skeletal muscle mass are largely responsible for the alterations seen at the functional level. This regulation is based on protein balance which controls anabolic and catabolic pathways in order to maintain protein content and optimal function (Sandri, 2008; Schiaffino, Dyar, Ciciliot, Blaauw, & Sandri, 2013). Both synthesis and degradation protein pathways are altered in muscle deconditioning situations, and it is well established that a dysregulation of their balance contributes to muscle atrophy (Chopard, Hillock, & Jasmin, 2009; Christopher S Fry & Rasmussen, 2011; Glass, 2005; Ventadour & Attaix, 2006).

Protein synthesis pathway

The PI3K-Akt-mTOR (Phosphoinositide 3 Kinase – Protein Kinase B – mammalian Target Of Rapamycin) axis constitutes the main signaling pathway whose activation by resistance training or protein ingestion guarantees the maintenance or increase of muscle mass (Bodine, Stitt, et al., 2001). Briefly, the activation of PI3K, stimulated by insulin or growth factors, increases the activity of Akt (also known as Protein Kinase B) (Rommel et al., 2001). This one plays an inhibitory role on TSC1/TSC2 complex by specific phosphorylations (Manning & Cantley, 2003). The interaction of mTOR with various proteins allows the formation of mTORC1 and mTORC2 complexes, which play a central role in the signaling cascade (Laplante & Sabatini, 2012). Final targets of mTOR as 4E-BP1, S6K1 or eukaryotic initiation factors (eIF3F, eIF2α) allow ribosomal biogenesis and protein translation (Holz, Ballif, Gygi, & Blenis, 2005).
In muscle deconditioning conditions, the diminution of synthesis flux is a usually observed phenomenon (Atherton et al., 2016). For example, Glover et al. (2008) showed that 14 days of joint immobilization were responsible for the decline in post-prandial synthesis flux. Body of evidence also demonstrated that inactivity in spaceflight and bedrest situations was able to reduce muscle and whole-body protein synthesis (Chopard, Hillock, et al., 2009; Ferrando et al., 1996; Ferrando, Paddon-Jones, & Wolfe, 2002; Stein, 1999). Animal studies using hindlimb suspension or denervation models also showed a decrease of synthesis flux after only few days of unloading (Bodine, Stitt, et al., 2001; A. V Gomes et al., 2012; Hornberger, Hunter, Kandarian, & Esser, 2001). These studies demonstrated that the downregulation of some mTOR pathway actors, such as Akt, S6K1 or eIF2-α, are implicated in muscle atrophy.
During aging, the loss of muscle mass and strength characteristic of sarcopenia is also associated with a decrease in protein synthesis. Indeed, multiple studies in rodents and humans reported lower rates of muscle protein synthesis in elderly and old rodents (P Balagopal, Rooyackers, Adey, Ades, & Nair, 1997; Christopher S Fry & Rasmussen, 2011; Fadia Haddad & Adams, 2006; Léger, Derave, De Bock, Hespel, & Russell, 2008; Paturi et al., 2010; S. Welle, Thornton, Jozefowicz, & Statt, 1993; Yarasheski, Welle, & Nair, 2002). More specifically, it has been reported a decrease in MyHC synthesis during aging, which was correlated with IGF-1 plasmatic concentrations, muscle mass, and strength (P Balagopal et al., 1997). This reduced MHC synthesis is at least due to a diminished transcription because RNA amounts of different isoforms are decreased with aging, particularly type IIa and IIx isoforms (Prabhakaran Balagopal, Schimke, Ades, Adey, & Nair, 2001; Short et al., 2005), but it is also well established that translational efficacy is lowered in the elderly (Fadia Haddad & Adams, 2006; Prod’homme et al., 2005).

Protein degradation pathways

There are four major pathways involved in muscle proteolysis: apoptosis, autophagy, the calpains and the ubiquitin-proteasome system. Each of these pathways and their implications in skeletal muscle deconditioning will be described in the following sections.

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Apotosis

Apoptosis, also known as “programmed cell death” is a process by which cells activate their own destruction in response to stresses. It can be activated by two types of pathways: an extrinsic one involving TNF-α (tumor necrosis factor α), and an intrinsic one involving mitochondria. The intrinsic axis acts through two intracellular signaling pathways dependent or not on caspases (cysteine-dependent aspartate-cleaving proteases) (E Marzetti, Calvani, Bernabei, & Leeuwenburgh, 2012). These enzymes permit the cleavage of numerous target proteins of nuclear envelope and DNA (Danial & Korsmeyer, 2004; Dupont-Versteegden, 2005). At the end of the process, cell proceeds to its auto-destruction and resultant fragments are eliminated by macrophages (Pollack, Phaneuf, Dirks, & Leeuwenburgh, 2002).
Although research on animal models suggests a key role of apoptosis in age-related muscle loss, evidence in humans is still lacking (Dirks & Leeuwenburgh, 2005; Dupont-Versteegden, 2005; Emanuele Marzetti & Leeuwenburgh, 2006; Siu, Pistilli, & Alway, 2005). The study of Whitman was the first to investigate age-related muscle apoptosis in humans. They reported an increase in the number of positive TUNEL cells but no changes in caspase-3/7, in vastus lateralis biopsies of older men (Whitman, Wacker, Richmond, & Godard, 2005a). Moreover, in a community-dwelling older adults, significant correlations were observed between caspase-dependent apoptotic signaling proteins and muscular thigh volume, as well as gait speed (Emanuele Marzetti et al., 2012).
Moreover, multiple studies of muscle disuse showed associations between loss of muscle CSA and the number of apoptotic cells within the tissue (Andrianjafiniony, Dupre-Aucouturier, Letexier, Couchoux, & Desplanches, 2010; Borisov & Carlson, 2000; Cheema, Herbst, McKenzie, & Aiken, 2015; Guo, Cheung, Yeung, Zhang, & Yeung, 2012; H. K. Smith, Maxwell, Martyn, & Bass, 2000). In these studies, apoptosis was measured by a variety of parameters, including caspase activation, mitochondrial EndoG release, or DNA fragmentation (involving the TUNEL staining method), and provide compelling data that apoptosis increases dramatically during the early phase of atrophy (Schwartz, 2019). Allen et al. (1997) reported for the first time an increase in apoptosis markers in atrophy induced by unloading conditions. The authors observed an elevation of apoptotic nuclei (measured via DNA fragmentation histochemical staining) in atrophied muscles of unloaded animals ; those results were confirmed later (Siu et al., 2005). However, the experiments of Bruusgaard research’s group pointed that skeletal muscle atrophy is not accompanied by myonuclear death (J. C. Bruusgaard et al., 2012; J. C. Bruusgaard, Johansen, Egner, Rana, & Gundersen, 2010; Jo C. Bruusgaard & Gundersen, 2008). Indeed, the recent review of Schwartz et al. (2019) underlined that despite the numerous studies reporting the presence of apoptotic nuclei within atrophied muscle tissue, these ones are not true myonuclei but rather condemned mononuclear cells that reside outside the muscle fiber.

Calpains system

The second system is regulated by calpains, whose functioning depends on calcium concentrations (Guroff, 1964). Muscular cells contain two types of calpains (type 1 and 2, also called µ-calpain and m-calpain). Cytoskeletal and membranous proteins, enzymes and transcription factors has been identified as potential substrates for calpains (E Dargelos, Poussard, Brule, Daury, & Cottin, 2008).
Abnormal increase of calpain activity have been showed in atrophic conditions like muscle disuse or denervation (Fareed et al., 2006; Huang, Zhu, & Zhu, 2016; Matsumoto, Fujita, Arakawa, Fujino, & Miki, 2014; Nelson, Smuder, Hudson, Talbert, & Powers, 2012). Matsumoto et al (2014) found an overexpression of calpain-2 in denervated and unloaded muscles of rats. Moreover, it has been suggested that the calcium-dependent proteolytic system is involved in sarcopenia. It is based on the overall increase in calpain activities with muscle aging and supported by the idea that calpain-mediated proteolysis of myofibrillar components strongly contribute to the loss of skeletal muscle mass and function with advanced age (Elise Dargelos et al., 2007; Samengo et al., 2012).

Table of contents :

INTRODUCTION
GENERAL INTRODUCTION
CHAPTER 1: SKELETAL MUSCLE DECONDITIONING
1.1. Generalities
1.2. Characteristics of muscle deconditioning
1.2.1. Loss of muscle strength
1.2.2. Loss of muscle mass
1.2.3. Myotyoplogic changes
1.2.4. Intermuscular adipose tissue accumulation
1.3. Dysregulation of protein balance: molecular pathways implicated
1.3.1. Protein synthesis pathway
1.3.2. Protein degradation pathways
1.4. Experimental models of muscle deconditioning
1.4.1. Cell models of muscle wasting
1.4.2. Animal models of muscle wasting
1.4.3. Human models of muscle wasting
1.5. Muscle deconditioning with aging
1.5.1. The frailty syndrome
1.5.2. Animal models of frailty
CHAPTER 2: THE REDOX BALANCE
2.1. Pro-oxidant molecules
2.1.1. Definition
2.1.2. Sources of free radicals
2.2. Antioxidants
2.2.1. Classification from a biochemical point of view
2.2.2. Classification from a cell physiology point of view
2.3. Concept of oxidative stress
2.3.1. Damage to DNA
2.3.2. Damage to lipids
2.3.3. Damage to proteins
2.4. The role of G6PD in oxidative stress protection
2.4.1. G6PD and the glutathione antioxidant system
2.4.2. Modulation of G6PD activity
2.5. Hypoactivity and redox regulation of muscle mass
2.5.1. Oxidative stress and protein synthesis pathway
2.5.2. Oxidative stress and proteolytic pathways
2.6. Redox balance and aging
2.6.1. Oxidative stress in old muscles
2.6.2. Oxidative stress and frailty
2.7. Antioxidant strategies against muscle deconditioning
2.7.1. Antioxidant strategies in aged muscles
2.7.2. Antioxidant strategies in inactive muscles
AIMS
STUDY I: G6PD OVEREXPRESSION: EFFECTS ON FRAILTY IN OLD MICE
MATERIAL AND METHODS
1. Experimental animals
1.1. Generation of a G6PD-transgenic mouse model
1.2. Animal care
1.3. Functional tests
1.4. The “Valencia score” of frailty
2. Biochemical analysis
2.1. Glutathione determination
2.2. Lipid peroxidation determination by HPLC
2.3. Carbonylated proteins determination
2.4. Western blotting
2.5. Antibodies
3. Histological analysis
4. RNA extraction and whole transcript analysis
5. Statistical analysis
RESULTS AND DISCUSSION
1. Longitudinal evaluation of frailty
1.1. Determination of frail mice for 5 functional parameters
1.2. The Valencia score of frailty
2. Oxidative stress parameters in skeletal muscles
2.1. Levels of glutathione in skeletal muscle
2.2. RONS-induced damage to lipids and proteins
3. Markers of skeletal muscle quality
3.1. Evaluation of skeletal muscle mass
3.2. Indicators of muscular “quality”
4. Transcriptomic analysis in skeletal muscle samples
4.1. Changes in transcriptomic profile
4.2. Differentially expressed genes involved in metabolic
pathways and biological processes
CONCLUSION
STUDY II: EFFECTS OF ANTIOXIDANT SUPPLEMENTATION ON HYPOACTIVITY-INDUCED MUSCLE DECONDITIONING
CONTEXT OF THE STUDY
MATERIAL AND METHODS
1. Overall study design
1.1 Subjects and ethics statement
1.2. Experimental protocol of HDBR
2. Measurements of maximal isometric voluntary contraction (MVC)
3. Muscle biopsies
4. Analysis of muscle samples
4.1. Cryosectionning and immunohistochemistry
4.2. Western-blotting
4.3. Antibodies
5. Statistical analysis
RESULTS AND DISCUSSION
1. Evaluation of muscle strength and muscle mass
1.1. Loss of muscle strength
1.2. Atrophy of muscle fibers
1.3. Changes in myofibers type distribution
2. Oxidative stress parameters
2.1. Markers of RONS-induced damage
2.2. Antioxidant enzymes
3. Mitochondrial parameters
3.1. Oxidative metabolism markers
3.2. Mitochondrial dynamics markers
4. Protein balance parameters
4.1. Protein synthesis pathway
4.2. Protein degradation pathway
4.3. Autophagy pathway
5. Parameters of adipogenesis
CONCLUSION
GENERAL CONCLUSION
AND PERSPECTIVES
RESUMEN EN ESPAÑOL
RESUME EN FRANCAIS
REFERENCES
ANNEXES

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