Journal of Signal Transduction Mitochondria are not the main and only source of ROS production in skeletal muscle during exercise. Indeed, other relevant sources of ROS production within muscle cells are nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) located within the sarcoplasmic reticulum, transverse tubules, and the sarcolemma [24, 25]. Phospholipase A2 (PLA2) is another known source of intracellular ROS production [17]. Arachidonic acid released from cell membranes by PLA2 is a substrate for ROS- generating enzyme systems such as the lipoxygenases [26]. Activation of PLA2 can stimulate NOXs [27] and increased PLA2 activity can promote ROS production in muscle mitochondria [28] and cytosol [29] and release ROS into the extracellular space [26]. Both calcium-dependent and -independent forms of PLA2 exist in skeletal muscle and both contribute to muscle ROS generation [17]. In particular, the calcium-independent isoforms are likely to be involved in cytosolic oxidant activity in skeletal muscle cells [29], whereas a 14-kDa calcium-dependent isoform located within mitochondria is reputed to stimulate mitochondrial ROS generation during contractile activity [30]. In this light it has been proposed [29] that the calcium-independent PLA2 is a major determinant of ROS activity under resting conditions, whereas during processes or stress elevating intracellular cal- cium concentration (contraction, inﬂammation, heat stress, etc.) the calcium-dependent PLA2 is activated to promote ROS production. Finally, superoxide anion is known to be generated by xanthine oxidase (XO) in the cytosol of contracting rat skeletal muscles cells [31]. However, human skeletal muscles contain lower levels of XO than rat muscle cells, and the question whether XO plays an important role in superoxide production in human skeletal muscle is still open [31, 32]. More ROS-generating mechanisms may be operative at the same time, as in the case of prolonged muscle ischemia where mitochondrial and cytoplasmic (via XO) ROS production has been simultaneously scored [33]. ROS can be generated through the above mechanisms not only within muscle cells but also in their proximity. For instance, during inﬂammation (a pathophysiological state that substantially alters cellular oxidative/antioxidant home- ostasis) inﬁltrated polymorphoneutrophils activate NOX producing ROS via the respiratory burst and many cytokines, which amplify in a feedforward cycle ROS production, are secreted within muscles [34]. For instance, during the early phase of muscle injury, inﬂammatory cytokines can bind to membrane receptors and activate speciﬁc ROS-generating enzymes, such as cyclooxygenase-2, NOX, and XO [35]. Endothelial cells from injured muscle are known to secrete TNF-α, interleukine (IL)-1, IL-6, and IL- 8, providing a positive feedforward cycle [36]. Whereas a transient oxidative stress is necessary in inﬂamed muscle cells to exert an antiseptic function and to activate various signal transduction pathways relevant to the restitutio ad integrum, prolonged severe oxidative stress may imbalance intracellular antioxidant homeostasis and hence long-term muscle welfare. The oxygen-centered species that mostly arises from the processes described so far is superoxide anion, but its 3 signiﬁcance in ROS signaling pathways seems to be limited to a role as a precursor signaling molecule. Indeed superoxide undergoes enzymatic or spontaneous dismutation, a process generating H2O2. H2O2 is a nonradical, is a weak oxidant with a relatively long half-life allowing its diﬀusion within cells and across cell membranes [37], reacts with many diﬀerent cellular molecules, and activates a wide number of signaling pathways. These properties render H2O2 the most relevant ROS signaling molecule in cells [38]. In contrast, H2O2 undergoing Fenton chemistry in the presence of redox active free iron ions or other transition metals can give rise to hydroxyl radicals, which react immediately with any surrounding biomolecules exerting most of the deleterious eﬀects associated with oxidative stress. In this light, iron homeostasis can be considered as a comodulator of ROS signaling and eﬀects. In particular, since skeletal muscle contains 10 to 15% of body iron—mainly in myoglobin and mitochondria—it could be particularly sensitive to alter- ations of iron homeostasis: accordingly it has been recently reported that levels of muscle nonheme iron and the iron transport protein, transferrin were elevated in senescence, suggesting that iron load is a signiﬁcant component of sarcopenia [39]. 3. Antioxidants and Modulation of Muscle Cells’ Sensitivity to ROS As it will be discussed throughout this review, the net eﬀect of ROS on cells’ signaling pathways and fate depends also on the cellular antioxidant capacity. The antioxidant network consists of enzymes, such as catalase, glutathione peroxidase (GPx), thioredoxin reductases (TRxs), superoxide dismu- tases (SODs), and soluble antioxidants such as glutathione (GSH) and vitamin E. Depending on its eﬃcacy, the antiox- idant cellular network plays a primary role in maintaining ROS below a physiologically compatible threshold level, thus allowing ROS to serve, theoretically, as signaling molecules and avoiding them to exert direct toxic eﬀects. Many antioxidant enzymes are known to be induced in response to increased ROS generation. The increased ROS ﬂux occurring in the course of strenuous exercise, through redox-sensitive mechanisms, induces the expression of γ-glutamylcysteinyl synthetase, the rate-limiting enzyme of GSH synthesis, of GPx, and of MnSOD [40]. Nuclear Factor KappaB (NF-κB), activator protein 1 (AP-1), and mitogen-activated protein kinases (MAPKs) have been identiﬁed as the major signaling pathways that can be activated by exercise-derived ROS and directly involved in the induction of the above antioxidant enzymes [40]. For instance, in the signaling path of MnSOD gene expression, NF-κB and AP-1 play an important role [41]: both NF-κB and AP-1 binding sites are present in the promoter of the mammalian MnSOD gene, and ROS have been shown to activate their binding. The above responses, whose extent might be genetically predetermined, represent a fundamental adaptive counter- measure to conditions potentially resulting in frank oxidative stress. A speciﬁc modulator of ROS activity, working in an “antioxidant-like” fashion in ROS-mediated autophagy and apoptosis (see also “ROS mediate autophagy and apoptosis”),