B) Representative Western-blot of Myogenin at cell confluence (C) and after 3 days of differentiation (D3). == Influence of SIRT3 down-regulation on mitochondrial activity == To address the effect of SIRT3 depletion on mitochondrial activity and biogenesis, we measured several parameters such as Candesartan (Atacand) respiratory ratio, enzymatic activities of the respiratory chain complexes involved in substrate oxidation, and ROS accumulation. In control KIAA0538 cells, the basal respiration rate significantly increased from the proliferation state to the third day of differentiation (Fig. overexpression; 2) a decrease in peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1) and citrate synthase protein expression reflecting an alteration of mitochondrial density; and 3) an increased production of reactive oxygen species (ROS) mirrored by the decreased activity of manganese superoxide dismutase (MnSOD). Altogether our data demonstrate that SIRT3 mainly regulates myoblast differentiation via its influence on mitochondrial activity. == Introduction == Skeletal muscle tissue is characterized by a high plasticity allowing tremendous metabolic adaptation in response to different physiological conditions. This flexibility occurs in parallel to changes in mitochondrial activity[1]. Recent studies have shown that mitochondria, besides their role in fuel metabolism, significantly influence muscle development, through the regulation of myoblast proliferation and differentiation, and the acquisition of contractile and metabolic features of muscle fibers[2],[3],[4],[5]. Indeed, mitochondrial activity controls myoblast Candesartan (Atacand) differentiation via the regulation of c-Myc, Myogenin and Calcineurin expression. The same molecular targets are involved in the inhibitory effect of chloramphenicol, an inhibitor of mitochondrial protein synthesis, on myogenic differentiation. Conversely, upregulation of mitochondrial activity upon overexpression of the mitochondrial triiodothyronine receptor (p43) stimulates terminal differentiation[2],[4],[5]. Among the metabolic regulators, the sirtuin family, composed of seven NAD+-dependent lysine deacetylases (NAD+: Nicotinamide Adenine Dinucleotide) is a group of metabolic sensors for cellular NAD+/NADH ratio. These proteins differ in tissue specificity, subcellular localization, enzymatic properties and targets[6]. Sirtuin1 (SIRT1), the most studied sirtuin, localizes to the nucleus where it deacetylates histones, transcription factors and their co-regulators. In muscle cells, SIRT1 interaction with MyoD and its co-activator P300/CBP-associated factor (PCAF) inhibits its function and prevents muscle differentiation[7]. Moreover, Fulco et al. reported that SIRT1 depletion, mediated by RNA interference, induces muscle cell differentiation in a non-permissive micro environment (AICAR insensitive)[8]. Three sirtuins are localized in mitochondria: SIRT3, SIRT4 and SIRT5, and participate in the regulation of ATP production, metabolism and cell signaling[9]. SIRT3 is considered as the major mitochondrial deacetylase since its depletion leads to mitochondrial protein hyperacetylation, an event not occurring after SIRT4 or SIRT5 inhibition[10]. In agreement with these observations, recent studies have established that, in addition to a weak deacetylase activity, SIRT4 and SIRT5 have other functions; Candesartan (Atacand) SIRT4 exerts an inhibitory ADP-ribosyl-transferase activity towards the glutamate dehydrogenase (GDH)[11]and SIRT5 was reported to exert a desuccinylase/demalonylase activity[12]. The first identified SIRT3 target was the mitochondrial protein acetyl-coenzyme A (acetyl CoA) synthase 2 (AceCS2) which requires deacetylation in order to Candesartan (Atacand) convert acetate to acetyl CoA in the presence of ATP[13],[14]. Similar positive effects are as well described upon SIRT3 dependent deacetylation of the glutamate dehydrogenase (GDH), an enzyme required for urea synthesis, and the long-chain acyl CoA dehydrogenase (LCAD), a central enzyme in the fatty acid oxidation pathway[15]. Moreover, SIRT3 modulates the production of cellular ROSviadeacetylation of antioxidant enzymes such as superoxide dismutase 2 (SOD2, MnSOD)[16],[17]. SIRT3 also controls ATP levels by modulating the activity of the respiratory chain complexes I and II upon binding to NDUFA9[18]and SdhA[19]subunits respectively. Consequently, it becomes increasingly clear that reversible lysine acetylation is a major post-translational modification of the mitochondrial proteome central for the maintenance of their proper function and for the adaptation of mitochondrial activity. In turn, our group previously described the involvement of mitochondrial activity in the regulation of myoblast differentiation and myogenic factor expression and/or activity. Since SIRT3 does Candesartan (Atacand) modulate mitochondrial activity, we have investigated here its influence on myoblast differentiation. == Materials and Methods == == Cell culture == Mouse myoblasts of the C2C12 cell line (ATCC) were grown in Dulbecco’s modified Eagle’s medium (DMEM) containing 4.5 g/l glucose, 0.584 g/l L-glutamine, and 3.7 g/l sodium bicarbonate and.