Journal of Signal Transduction upon ROS accumulation exhibits an increased conductance associated with MPTP opening and dissipation of ΔΨ, thus favouring the eﬄux of apoptotic proteins located in the intermembrane space and ﬁnally cell death [107]. Notably, the pro- and antiapoptotic Bcl2-family proteins are released via VDAC1 action and ROS may further aﬀect these responses as they are known, in nonmuscle cells, to down-regulate the endogenous levels of the antiapoptotic protein Bcl-2 [108]. The mechanism through which Bcl-2 levels are aﬀected by ROS has been studied by Azad et al. in nonmuscle cell types and seems to depend on superoxide anion-related degradation of Bcl-2 protein through the ubiquitin-proteasomal pathway [109]. Furthermore, under oxidative stressing conditions, ROS activate a signaling cascade involving the protein kinase C (PKC) b-dependent phosphorylation of the Shc adaptor protein p66shc and its translocation to the mitochondrial matrix. In particular, the mitochondrially translocated frac- tion of p66shc behaves as redox enzyme that utilizes reducing equivalents derived from the mitochondrial electron trans- port chain to produce H2O2 in the intermembrane space, an event which is known to trigger apoptosis [110, 111]. The accumulation of ROS within the mitochondrial matrix, as well as their capacity of triggering apopto- sis, is counteracted/regulated by mitochondrial antioxidant enzymes, namely, phospholipids hydroperoxide glutathione peroxidase, GPx, and Mn-SOD [3, 112]. Thus increased mitochondrial production of ROS is involved at multiple levels in promoting apoptosis in skeletal muscle cells, an event which participates in the aetiology and progression of numerous pathologies including sarcopenia and disuse muscle atrophy as well as in aging [71, 113]. Physical training and exercise are known to increase mitochondrial biogenesis and density as well as mitochon- drial ROS production especially during repeated contrac- tions [85]. Therefore and unless other determinants are considered, it might appear paradoxical that although a routine of regular exercise is associated with numerous health beneﬁts, physical exercise might potentially promote oxidative stress and ROS-associated apoptosis of skeletal muscle cells [17]. Indeed, chronic contractile activity (CCA) and endurance training induce an adaptive response in skeletal muscle cells leading to increased mitochondrial biogenesis [114] and—theoretically—an obligatory increase in a number of proapoptotic mitochondrial proteins and byproducts such as ROS. However, as a matter of fact recent evidence indicates that mitochondria isolated from rat skeletal muscle subjected to CCA seem to acquire an anti- apoptotic, rather than proapoptotic, behaviour [114]. The study also addressed the problem of the relative antiapoptotic role acquired by diﬀerent mitochondrial subpopulations from CCA-trained muscles, namely, the intramyoﬁbrillar (IMF) and the subsarcolemmal (SS) mitochondria. The release of both Cyt C and AIF caused by exogenous H2O2 from CCA-isolated IMF and SS mitochondria was decreased; CCA augmented the expression of antiapoptotic HSP70 and caspase recruitment domain protein in either SS or IMF and caused a decreased ROS generation in IMF mitochondria. On the contrary, states III and IV respiring SS mitochondria 7 showed a modestly increased rate of ROS generation as well as an increased resilience of MPTP opening. It was then hypothesized that these eﬀects might collectively reﬂect the overall reduced apoptogenic capacity acquired by mitochon- dria following CCA training of skeletal muscles and that, in particular, the slight increase of ROS generated by SS would contribute to the activation of redox-sensitive transcription factors promoting muscle ﬁber plasticity and adaptation, rather than to function as proapoptotic triggers. Again, such a scenario is indicative of the diverging eﬀects that ROS may assume depending on speciﬁc situations of cells’ life, rather than on their net concentration and site of generation. 6. ROS Signaling and Myogenic Differentiation Increasing evidence indicates that ROS are capable of aﬀecting—mostly reducing—the eﬃciency of myogenic dif- ferentiation. The integrity/alteration of myogenic diﬀer- entiation is central to many physiological and pathologi- cal processes. Successful diﬀerentiation of satellite-derived myoblasts into functioning and integrated myotubes is a fundamental prerequisite for muscle regeneration, a repair process which is of primary importance in maintaining muscle function [115]. Notably, oxidative stress is known to play a concausal and detrimental role in a variety of multifactorial muscular pathologies characterized by prolif- eration/diﬀerentiation imbalance such as Duchenne dystro- phy [116], myotonic dystrophy [117], sarcopenia [118], and cachexia [119]. The role of ROS in this context has been extensively documented. Ardite et al. [120] showed that ROS induced a strong depletion of the intracellular GSH pool: notably depletion of GSH causes further intracellular accumulation of ROS which favors NF-κB activation, thus contributing to the lower expression of MyoD and impaired myogenesis (see below). According to Ardite et al. [120], data from our group [5] indicate that a mildly toxic H2O2 treatment during the early stages of C2C12 myoblast diﬀerentiation results in GSH depletion and strongly impairs the diﬀerentiative outcome. This eﬀect is unlikely to be a mere result of ROS-induced cell demise: indeed, the cells surviving H2O2, although exhibiting a partial and late recovery of protein synthesis and of viability, were unable to continue and execute the diﬀerentiative task. These cells also displayed a strong and long-lasting reduction of the mRNA levels of MyoD, which is involved in early stem cell commitment, and of myogenin and MRF4, both recruited at later diﬀerentiation times [121, 122]. Whether the transcription of these muscle regulatory factors (MRFs) is a result of a speciﬁc signaling promoted by ROS or of a cell suﬀering is still to be understood. Under the same conditions depressing these MRFs, insulin- like growth factor 1 (IGF-1) which plays a pivotal role in controlling muscle growth [123], was inhibited to an even greater extent (see also “ROS and IGF-1 signaling”). Interestingly, H2O2-injured cells showed signs of extensive mitochondrial degeneration (swelling and disruption) and lower mitochondrial density, suggesting that these organelles