8 are speciﬁcally targeted by—or particularly sensitive to— exogenous ROS. Loss of mitochondria is a clearly detrimental event in a process typically requiring active mitochondrio- genesis such as muscle diﬀerentiation [4, 124]. ROS generated by the inﬂammatory cytokine TNFα are known to inhibit myogenesis, and this eﬀect is widely attributed to oxidative activation of NF-κB and subsequent gene expression [125–127]. However, the eﬀect of TNFα is likely to be more complex since Langen et al. [9] showed that TNFα causes loss of myogenic capacity of C2C12 cells via NF-κB-dependent and-independent and oxidative-sensitive and-insensitive pathways. In particular they hypothesized that an oxidative-sensitive, NF-κB-independent mechanism might involve the blockage of the formation of functional catenin-adherin complexes proximate to the cell membrane [128]. Potentially, disruption of these complexes and the resulting alteration of cell-matrix and cell-cell interactions, might be responsible for the inhibition of myotube forma- tion independently of NF-κB. The redox regulation of the NF-κB family of tran- scriptional activators plays a central role in diﬀerentia- tion, adaptation, and death of muscle cells. This role is extremely complex: indeed the eﬀects promoted by NF-κB are sometimes contradicting. As an example, although ROS can directly stimulate NF-κB, oxidized NF-κB has a dimin- ished DNA-binding activity [17]. NF-κB has been mostly associated with a negative regulation of skeletal muscle diﬀerentiation [119, 129, 130]. NF-κB is constitutively active in proliferating myoblasts and can inhibit myogenesis by promoting a mitogenic activity via cyclin D1 or by inhibiting the synthesis of MyoD, a muscle-speciﬁc helix-loop-helix transcription factor operating in muscle development and repair [131–133]. More recently, NF-κB was shown to sup- press myoﬁbrillar gene expression through the regulation of the myogenic transcriptional repressor Yin Yiang 1 [134]. Moreover, treatment of primary myoblasts with the NF- κB inhibitor curcumin stimulates myoblast fusion thereby enhancing myogenesis and repair [125]. In line with these in vitro ﬁndings, activation of the TNFα pathway by muscle gene transfer inhibits regeneration in vivo, while muscle- speciﬁc deletion of the heteromeric kinase complex IKK was recently described to promote secondary myogenesis in response to acute injury signals [126, 127]. Activation of NF-κB downstream ROS formation is also capable of stimulating the activity of inducible nitric oxide synthase (iNOS), whose role in myogenic process is controversial [24]. Some authors found that iNOS activity suppresses muscle diﬀerentiation, whereas others reported that stimulation of iNOS via NF-κB represents a positive and necessary stimulus for muscle diﬀerentiation, that iNOS activity paralleled myogenesis from the early to later stages in H9C cells and that ROS formed by NOX 2 were the basic trigger leading to iNOS stimulation via NF-κB recruitment [24, 135, 136]. Blockage of this pathway, or inhibition of iNOS with speciﬁc inhibitors, led to diﬀerentiative arrest. Also, a recent article by Lee et al. [137] indicates that complex-I-derived superoxide anions, produced through reverse electron transport, were dismutated into H2O2 by MnSOD induced via NF-κB activation and that H2O2 stimulated muscle diﬀerentiation Journal of Signal Transduction as a signaling messenger. Thus the scenario arising from these results would indicate that ROS negatively or positively regulate muscle diﬀerentiation via the signaling pathways involving NF-κB activation. Another evidence which lends support to the detrimental role of ROS in muscle diﬀerentiation comes from the studies on the role of p66Shc in skeletal muscle ischemic injury. p66Shc, along with its isoforms p46 and p52, constitutes the mammalian Shc adaptor protein group. The three isoforms share a common structure, but p66ShcA has the unique feature of an additional domain at the N terminus which contains a serine residue at position 36 (Ser-36) that is phosphorylated in response to several stimuli, including H2O2. Due to this feature p66 isoform regulates ROS metabolism and apoptosis [138, 139]; indeed, a fraction of p66ShcA is localized in the mitochondria where, as discussed above, it produces mitochondrial ROS as signaling molecules for apoptosis [110, 111]. Interestingly, both p66ShcA KO cells and mice display lower levels of intracellular ROS [139– 141] and are less prone to apoptosis induced by an array of diﬀerent stimuli. Also, p66Shc KO mice are resistant to ischemia-induced apoptosis and show decreased muscle damage in response to hind limb ischemia [142]. More recently, Zaccagnini et al. [143] unravelled the role of p66Shc and ROS in muscular damage and regeneration following acute hind limb ischemia in both WT and p66Shc KO mice. WT mice showed detectable levels of oxidative stress markers during the postischemic and regenerative stages; on the contrary, the same markers were undetectable in KO mice. More interestingly, although the initial ischemic damage was identical and no advantage in terms of muscle vascularization and perfusion was observed in KO mice, their regenerative capacity was signiﬁcantly higher as compared to WT. Satellite cell populations were similar in both groups, but those from KO mice showed a higher proliferation rate at ﬁrst and spontaneous diﬀerentiation when cultured under prodiﬀerentiative conditions. Finally, p66Shc KO satellite cells were resistant to the myogenic inhibition induced by H2O2 acute challenge or hypoxia. The authors proposed diﬀerent and possible explanations for the above eﬀects. The ﬁrst one involves the diﬀerent availability of NO— whose promyogenic role has been discussed above—in KO mice: since active p66Shc generates superoxide anions, which consume available NO forming the toxic species peroxynitrite, p66Shc KO mice would beneﬁt of higher NO availability and would not suﬀer of peroxynitrite toxicity, two eﬀects favouring myogenesis and muscle regeneration. Another plausible mechanism involves the NAD+-dependent histone deacetylase Sir2. Sir2 deacetylase activity is depen- dent on the ﬂuctuation of cytosolic NAD+/NADH ratio, that is, the cellular redox state [144]. Under conditions of high ROS concentrations, NAD levels increase and promote Sir2 activation, which in turn inhibits MyoD-dependent transcription. p66Shc KO mice are characterized by lower levels of ROS and, as a result, decreased Sir2 activity, that is a condition which aﬀects MyoD functions to a lesser extent. Finally, since oxidative DNA damage may trigger a diﬀer- entiation checkpoint and cause a reversible inhibition of myogenic diﬀerentiation targeting MyoD phosphorylation,