**2. REDOX Imbalance as a Mechanism of Muscle Wasting**

Skeletal muscle atrophy is a process that occurs as a result of conditions such as disuse, malnutrition, aging and in certain states of disease. Nonetheless, it is characterized firstly by a decrease in muscle mass (and volume), force production and, on a more detailed perspective, by a diminishment of protein content and fibre diameter [35]. Moreover, the primary loss in muscle strength that occurs with atrophy results from the rapid destruction of myofibrils, the contractile machinery of the muscle, constituting around >70% of the muscle protein [36].

Among all the potential aetiological foundations of muscle wasting, reactive oxygen species (ROS) generation, including the oxidative damage and/or the defective redox signalling, has stood out as the possible main explanation [37–39].

ROS are reactive molecules that contain oxygen, and this family is comprised of free radicals (i.e., species with at least one unpaired electron) and nonradical oxidants (i.e., species with their electronic ground state complete). The chemical reactivity of the various ROS molecules is vastly different; for instance, hydroxyl (•OH), the most unstable, reacts immediately upon formation with biomolecules in its vicinities, whereas hydrogen peroxide (H2O2) is capable of crossing cell membranes to exert its effects beyond its original compartment [40–42] (Table 1).


**Table 1.** The most common reactive oxygen species, antioxidants and respective scavenging reactions.

ROS are generated by various sources, mainly endogenous sources, including mitochondrial respiratory chain enzyme, nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity, microsomal cytochrome P450 and xanthine oxidase; and exogenous sources such as ultraviolet radiation, X- and gamma (γ)-rays, ultrasounds, pesticides, herbicides, and xenobiotics [43]. Superoxide anion (O2- •) is the most frequently generated radical, under physiological conditions. Its main source is the inner mitochondrial membrane, in the complexes I and III, during respiratory chain, by the inevitable electron leakage to O2 [44,45]. It can also be generated in the short transport chain of endoplasmic reticulum upon electron leakage and during NOX activity, by transferring one electron from nicotinamide adenine dinucleotide phosphate (NADPH) to O2 [46].

To cope with ROS, the cells have developed control systems to regulate oxidation/reduction balance, since redox balance is critical. A key component is the antioxidant system, which prevents ROS accumulation and deleterious actions. The cells contain both enzymatic and non-enzymatic antioxidants that work by mitigating ROS effects and by drastically delaying/preventing oxidation from happening. Key enzymatic antioxidants are superoxide dismutase (SOD), catalase, glutathione peroxidase (GPx) and thioredoxin (Trx), whereas

non-enzymatic are mainly vitamin C (ascorbic acid) and E (tocopherol), zinc and selenium, glutathione, plant polyphenols and carotenoids [47,48]. These act primarily by using three different strategies: (1) scavenging ROS; (2) converting ROS molecules into less reactive ones, and (3) chelation via metal binding proteins. Throughout the cells, antioxidants are compartmentalized in both organelles and cytoplasm, but also exist in the interstitial fluid and blood [49].

ROS are normal products of cell metabolism with significant physiological roles. They regulate signalling pathways (redox signalling) by changing the activity of structural proteins, transcription factors, membrane receptors, ion channels and protein kinases/phosphatases [50,51]. ROS physiological roles depend partly on antioxidant control, establishing a redox balance. When redox homeostasis is disrupted, due to the rising of ROS levels and the unlikely neutralization by the antioxidant defence, a state referred to as oxidative stress (OS) occurs. This leads to an impairment of redox signalling and induces molecular damage to biomolecules [52,53]. Moreover, OS has a graded response, with minor or moderated changes provoking an adaptive response and homeostasis restoration, whereas violent perturbations lead to pathological insults, damage beyond repair and may even lead to cell death [53]. Interestingly, something that is not appreciated often is that our understanding of "low" or "high" response regarding ROS levels is somewhat imprecise, redox time-courses in vivo are scarce and our knowledge is based of immunohistochemical analysis or measuring more stable elements of the family [54,55].

As in other tissues, redox signalling in skeletal muscle has important roles, being the base of skeletal muscle function to elicit exercise adaptation. It supports the neuromuscular development and the long-term remodelling/adaptation of contractile activity [56,57]. Moreover, regulated ROS levels are also involved in skeletal muscle regeneration, regulating the activity of skeletal muscle stem cells, through redox-sensitive signalling pathways [58] (Figure 2).

When an ROS overproduction occurs, cells are capable of maintaining a redox state by activating distinct transcription factors that induce the transcription of antioxidant enzymes to tilt the balance back to homeostasis, protecting them from OS [59,60]. One important transcription factor is the nuclear factor erythroid 2-related factor 2 (Nrf2), which is a ubiquitous protein that modulates OS [61]. In response to elevated ROS levels, Nrf2 triggers the expression of NADPH quinone oxidoreductase (NQO1), heme oxygenease-1 (HO-1), glutamate-cysteine ligase catalytic (GCLC) and glutamate-cysteine ligase modifier (GCLM), which are enzymes involved in redox homeostasis maintenance, cellular defence and detoxification [62,63]. Moreover, enzymes that encapsulate the redox cycling group, mediating the elimination of ROS such as thioredeoxin, thioredoxin reductase, sulfiredoxin, peroxiredoxin, gluthatione peroxidase, superoxide dismutase 1 (SOD1), catalase and various glutathione S-transferases, are all of them targeted by Nrf2 [64].

However, during ageing, cells produce even more ROS, mainly from mitochondria and NOX, and even though the activity of antioxidant enzymes in cells and muscle also increases with age, this compensatory adaptation is not sufficient to neutralize ROS levels [37–39]. These increased ROS levels cause deleterious macromolecules oxidative modification, leading not only to various cellular dysfunctions, but also affecting signal transduction pathways that control multiple essential cellular processes, such as protein turnover, mitochondrial homeostasis, energy metabolism, antioxidant gene expression and redox balance (see, for example, [65] for more details). Moreover, the systemic increase in ROS, associated with an OS state, increases proinflammatory transcription factors levels, for instance, nuclear factor kappa B (NF-kB) [66,67]. NF-kB regulates specific UPS genes and leads to the expression of proinflammatory cytokines such as IL-6 and TNF-α that are involved in the development of muscle atrophy [68–70].

**Figure 2.** Diagram of the skeletal muscle microenvironment. This niche is composed of various cell types and ECM proteins. In adult skeletal muscle, the quiescent satellite cells stand on the myofiber, under the basal lamina, being surrounded by the ECM, containing blood vessels, nerves, immune cells, fibro-adipogenic progenitors (FAPs), adipocytes and myofibroblast. The satellite cell states are regulated by their interactions with the surrounding microenvironment, direct interaction (e.g., M-cadherin) between muscle fibres and satellite cells; or interact with a variety of components of the ECM and cytokines and growth factors. In addition, stromal cells present can physically interact with satellite cells and release cytokines, growth factors and ECM components, which influence the behaviour of satellite cells, contributing to muscle growth, homeostasis and regeneration.

In summary, ROS load increment and the establishment of an OS state are detrimental to muscle function and are associated with the mechanism of skeletal muscle atrophy [71].

There are two common but distinct conditions that are characterized by skeletal muscle loss, which are sarcopenia and cachexia. In sarcopenia, skeletal muscle loss occurs in a slow and progressive way, being associated with ageing process (in the absence of disease), whereas, in cachexia, skeletal muscle loss is associated with inflammatory conditions (e.g., AIDS and sepsis) and chronic diseases such as cancer, diabetes, obesity, chronic obstructive pulmonary disease, chronic heart failure, chronic liver disease and chronic kidney disease [72–74].
