Journal of Signal Transduction suggest that the widely reported increase in antioxidant enzyme activities in aging skeletal muscle do not depend on enhanced gene transcription, but can rather derive from translational and/or posttranslational mechanisms. Since aged skeletal muscles are aﬀected by augmented levels of lipid peroxidation, protein oxidation, and DNA damage, these compensatory increases in antioxidant enzyme activity are ineﬀective in counteracting increased ROS generation. 4. ROS, Mitochondrial Biogenesis and Function It is well known that oxidative signals aﬀect mitochondrial biogenesis, morphology, and function in skeletal muscle cells [69–71]: again, the eﬀect of ROS seems to be bifaceted and controversial. Indeed, ROS may be important either in eliciting pathological eﬀects leading to mitochondrial dysfunction and cell death ([72, 73], see also below “Mitochondrial ROS Mediate Autophagy and Apoptosis”), or play physiological roles promoting positive responses in mitochondrial biogenesis and function. Mitochondrial bio- genesis is dependent on the expression of the mitochondrial genome and the nuclear genes that encode mitochondrial proteins [74]. An important pathway triggered by ROS is that leading to the upregulation of the mitochondrial biogenesis master gene PGC-1α. The PGC-1α transcriptional coactivator is a major regulator of energy metabolism [75]. It controls many aspects of oxidative metabolism, including mitochondrial adaptations, insulin-sensitizing via the upregulation of selected genes involved in fatty acid β- oxidation, glucose transport, and oxidative phosphorilation [76–79]. The mitochondrial biogenesis signaling activated by PGC-1 members family involves the transcription factors that regulate expression of nuclear genes such as nuclear respiratory factor (NRF) 1/2 and estrogen-related receptor- α (ERR-α). These three latter genes control the expression of nuclear genes encoding mitochondrial proteins and induce expression of mitochondrial transcription factor A (T- fam), which regulates mtDNA replication and transcription, thus activating the coordinated expression of mitochondrial proteins [80, 81]. Several signaling kinases have been involved in medi- ating PGC-1α transcriptional activation in response to a variety of stimuli among which the most important are calcium/calmodulin-dependent protein kinase (CaMK) type IV [82], AMPK [83], and p38 mitogen-activated protein kinase [84]. Their activation induces the PGC-1α promoter transcriptional regulation [69]. Recently, it has been demon- strated that mitochondrial biogenesis in skeletal muscle is controlled, at least in part, by a redox-sensitive mechanism and that physical exercise, increasing the ROS production over the physiological level, stimulates the muscle PGC- 1α/NRF-1/T-fam signaling [85]. Irrcher and colleagues have evaluated the link between ROS levels and PGC-1α gene expression [69] in C2C12 cells. They found that endoge- nously produced ROS, at least within skeletal muscle cells, are important for the maintenance of PGC-1α expression levels within a normal physiological range. Indeed, quenching basal endogenous ROS with N-acetylcysteine (NAC) results in reduced PGC-1α mRNA expression, an eﬀect which is 5 unrelated to any inhibition of PGC-1α promoter activity, but probably dependent on the enhanced instability of PGC-1α mRNA occurring in a low ROS environment. On the contrary, increasing ROS levels with exogenous H2O2 augments PGC-1α transcription indirectly via the AMPK activation caused by the oxidatively-induced ATP depletion. This stimulates the binding of USF-1 to an Ebox within the PGC-1α promoter, increases transcription and results in the induction of PGC-1α mRNA expression, whose stability would also be restored in a more ROS-rich environment. The interplay of PGC-1α and ROS is further strengthened by the fact that, besides being a key modulator of mitochondrial biogenesis, it is important in regulating the expression level of protective enzymes acting against ROS generation and damage [86]. Indeed, experiments with either genetic knockouts (KOs) or using RNA interference for PGC1α show that the ability of ROS to induce a ROS-scavenging program depends largely on PGC-1α activity [86]. This response includes genes encoding for antioxidant enzymes localized either within mitochondria (MnSOD) or cytosol (catalase and GPxs). Indeed, cells lacking PGC-1α are more susceptible to the toxicity induced by oxidative stress caused by H2O2 [86]. These latter eﬀects of PGC-1α are likely to represent a compensatory response where it plays a central role in the adaptation of cellular energy metabolism, mito- chondrial biogenesis and antioxidant capacity in response to oxidative challenge. At this regard and extending previous research from our group [5], we have recently addressed the problem of the role of PGC-1α in C2C12 myoblasts subjected to oxidative stress during the early stages of diﬀerentiation. In particular, we examined the eﬀect of a mildly toxic concentration of exogenously added H2O2 [0.3 mM] on the regulation of PGC-1α expression and its relationship with AMPK activation (unpublished observa- tions). According to Kang and Irrcher [69, 85], we found that 1 h treatment with H2O2 markedly increased PGC-1α mRNA expression. It is of worth that, concurrently, we also found an increased phosphorylation of AMPK as compared to untreated cells, suggesting that oxidative stress induces PGC-1α through the AMPK signaling pathway. However, despite the fact that challenged C2C12 myoblasts rapidly activate a defense-oriented signaling cascade, they displayed a 30–40% reduction of their viability as well as a survivors’ reduced diﬀerentiative eﬃciency during the post-challenge incubation stage (up to 7 days of culture). This observation would imply that, besides probably being an obligatory and physiological response to ROS, activation of AMPK and of PGC-1α may not be suﬃcient to aﬀord a complete protection to cells against an overwhelming oxidative stress. Accumulating or excessive oxidative stress is known to be detrimental for mitochondria: for instance mtDNA repre- sents a critical target for oxidative damage [49]. Indeed, mtDNA mutations are known as being an etiological factor in oxidative stress-related disorders including cardiovascular diseases and inherited or acquired neurodegenerative dis- orders, mitochondrial myopathies, and the normal aging process.