Sir2 (silent information regulator 2) is an NAD ؉ -dependent histone deacetylase that contributes to longevity in yeast. SIRT1, a mammalian Sir2 ortholog, deacetylates histones and various transcription factors, including p53, FOXO proteins, and peroxisome proliferator-activated receptor-␥. We found that its subcellular localization varied in different tissues of the adult mouse. Some subsets of neurons predominantly expressed SIRT1 in the cytoplasm, but ependymal cells expressed it in both the nucleus and cytoplasm. On the other hand, spermatocytes expressed SIRT1 only in the nucleus. Cardiomyocytes in the day 12.5 mouse embryo expressed SIRT1 exclusively in the nucleus, but in the adult heart, they expressed it in both the cytoplasm and nucleus. C2C12 myoblast cells expressed SIRT1 in the nucleus, but it localized to the cytoplasm after differentiation. LY294002, an inhibitor of phosphoinositide 3-hydroxykinase, strongly inhibited the nuclear localization of SIRT1 in undifferentiated C2C12 cells. In a heterokaryon assay, SIRT1 shuttled between the nucleus and cytoplasm, and leptomycin B, an inhibitor of CRM1-mediated nuclear exportation, inhibited this shuttling. Two nuclear localization signals and two nuclear export signals were identified by deletion and site-directed mutation analyses. Overexpressed nuclear (but not cytoplasmic or dominant-negative) SIRT1 enhanced the deacetylation of histone H3 in C2C12 cells. Moreover, only the nuclear form suppressed the apoptosis of C2C12 cells induced by antimycin A, an oxidative stressor. These findings indicate that nucleocytoplasmic shuttling is a novel regulatory mechanism of SIRT1, which may participate in differentiation and in inhibition of cell death.The Sir2 (silent information regulator 2) proteins are a family of class III histone deacetylases found in organisms from bacteria to humans (1). Unlike class I and II histone deacetylases, the Sir2 family requires the cofactor NAD ϩ for catalytic activity (2). In yeast, Sir2 participates in heterochromatic silencing at mating-type loci (3). Sir2 extends the life span of yeast by suppressing recombination in the rDNA region and consequently reducing the formation of extrachromosomal rDNA circles (4), a cause of senescence (5). Caloric restriction extends the life span in organisms ranging from yeast to mammals, and the Sir2 family plays an essential role in this effect (6, 7).In mammals, there are seven members of the Sir2 family, termed sirtuins, of which SIRT1 is the closest homolog of yeast Sir2. SIRT1 regulates metabolic responses in adipocytes and liver. It promotes fat mobilization in white adipocytes by repressing peroxisome proliferator-activated receptor-␥ (8). SIRT1 deacetylates the transcription coactivator PGC1␣, thereby inducing the expression of gluconeogenic genes and the repression of glycolytic genes (9). SIRT1 contributes to cell survival by deacetylating and thereby repressing the activity of the tumor suppressor p53 (10 -12). The transcriptional activation of stress-resistance genes by FOXO pro...

Oxidative stress plays a pivotal role in chronic heart failure. SIRT1, an NAD ؉ -dependent histone/protein deacetylase, promotes cell survival under oxidative stress when it is expressed in the nucleus. However, adult cardiomyocytes predominantly express SIRT1 in the cytoplasm, and its function has not been elucidated. The purpose of this study was to investigate the functional role of SIRT1 in the heart and the potential use of SIRT1 in therapy for heart failure. We investigated the subcellular localization of SIRT1 in cardiomyocytes and its impact on cell survival. SIRT1 accumulated in the nucleus of cardiomyocytes in the failing hearts of TO-2 hamsters, postmyocardial infarction rats, and a dilated cardiomyopathy patient but not in control healthy hearts. Nuclear but not cytoplasmic SIRT1-induced manganese superoxide dismutase (Mn-SOD), which was further enhanced by resveratrol, and increased the resistance of C2C12 myoblasts to oxidative stress. Resveratrol's enhancement of Mn-SOD levels depended on the level of nuclear SIRT1, and it suppressed the cell death induced by antimycin A or angiotensin II. The cell-protective effects of nuclear SIRT1 or resveratrol were canceled by the Mn-SOD small interfering RNA or SIRT1 small interfering RNA. The oral administration of resveratrol to TO-2 hamsters increased Mn-SOD levels in cardiomyocytes, suppressed fibrosis, preserved cardiac function, and significantly improved survival. Thus, Mn-SOD induced by resveratrol via nuclear SIRT1 reduced oxidative stress and participated in cardiomyocyte protection. SIRT1 activators such as resveratrol could be novel therapeutic tools for the treatment of chronic heart failure.Heart failure arises as a consequence of various heart diseases, including myocardial infarction, hypertension, and idiopathic dilated cardiomyopathy (DCM).2 The death of cardiomyocytes and the consequent maladaptive changes in the remaining myocytes and extracellular matrix induce the clinical manifestation of heart failure (1). Over the past 20 years, the arsenal of treatments available for heart failure has increased considerably, with the introduction of ␤ blockers, angiotensin-converting enzyme inhibitors, angiotensin II type 1 receptor blockers, aldosterone antagonists, and nonpharmacological therapies including cardiac resynchronization therapy (2). However, even with the very best current therapy, the annual mortality rate among patients with heart failure is still ϳ10% (3).The mitochondrial electron transport chain is the main source of reactive oxygen species (ROS) in most cells (4). Hearts consume large amounts of O 2 and yield high levels of ROS (5). Various factors, including angiotensin II and tumor necrosis factor-␣, also induce ROS formation, leading to cardiomyocyte death and heart failure (5). Superoxide dismutase (SOD) has a pivotal role in the detoxification of ROS. SOD catalyzes the dismutation of superoxide into oxygen and hydrogen peroxide, which in turn is reduced to water by catalase and glutathione peroxidase (5). Three isoforms of ...

Neural precursor cells (NPCs) differentiate into neurons, astrocytes, and oligodendrocytes in response to intrinsic and extrinsic changes. Notch signals maintain undifferentiated NPCs, but the mechanisms underlying the neuronal differentiation are largely unknown. We show that SIRT1, an NAD ؉ -dependent histone deacetylase, modulates neuronal differentiation. SIRT1 was found in the cytoplasm of embryonic and adult NPCs and was transiently localized in the nucleus in response to differentiation stimulus. SIRT1 started to translocate into the nucleus within 10 min after the transfer of NPCs into differentiation conditions, stayed in the nucleus, and then gradually retranslocated to the cytoplasm after several hours. The number of neurospheres that generated Tuj1 ؉ neurons was significantly decreased by pharmacological inhibitors of SIRT1, dominant-negative SIRT1 and SIRT1-siRNA, whereas overexpression of SIRT1, but not that of cytoplasm-localized mutant SIRT1, enhanced neuronal differentiation and decreased Hes1 expression. Expression of SIRT1-siRNA impaired neuronal differentiation and migration of NPCs into the cortical plate in the embryonic brain. Nuclear receptor corepressor (N-CoR), which has been reported to bind SIRT1, promoted neuronal differentiation and synergistically increased the number of Tuj1 ؉ neurons with SIRT1, and both bound the Hes1 promoter region in differentiating NPCs. Hes1 transactivation by Notch1 was inhibited by SIRT1 and/or N-CoR. Our study indicated that SIRT1 is a player of repressing Notch1-Hes1 signaling pathway, and its transient translocation into the nucleus may have a role in the differentiation of NPCs.

Muscular dystrophies are inherited myogenic disorders accompanied by progressive skeletal muscle weakness and degeneration. We previously showed that resveratrol (3,5,4Ј-trihydroxy-trans-stilbene), an antioxidant and activator of the NAD ϩ -dependent protein deacetylase SIRT1, delays the progression of heart failure and prolongs the lifespan of ␦-sarcoglycan-deficient hamsters. Because a defect of dystroglycan complex causes muscular dystrophies, and ␦-sarcoglycan is a component of this complex, we hypothesized that resveratrol might be a new therapeutic tool for muscular dystrophies. Here, we examined resveratrol's effect in mdx mice, an animal model of Duchenne muscular dystrophy. mdx mice that received resveratrol in the diet for 32 weeks (4 g/kg diet) showed significantly less muscle mass loss and nonmuscle interstitial tissue in the biceps femoris compared with mdx mice fed a control diet. In the muscles of these mice, resveratrol significantly decreased oxidative damage shown by the immunostaining of nitrotyrosine and 8-hydroxy-2Ј-deoxyguanosine and suppressed the up-regulation of NADPH oxidase subunits Nox4, Duox1, and p47 phox . Resveratrol also reduced the number of ␣-smooth muscle actin (␣-SMA) ϩ myofibroblast cells and endomysial fibrosis in the biceps femoris, although the infiltration of CD45ϩ inflammatory cells and increase in transforming growth factor-␤1 (TGF-␤1) were still observed. In C2C12 myoblast cells, resveratrol pretreatment suppressed the TGF-␤1-induced increase in reactive oxygen species, fibronectin production, and expression of ␣-SMA, and SIRT1 knockdown blocked these inhibitory effects. SIRT1 small interfering RNA also increased the expression of Nox4, p47 phox , and ␣-SMA in C2C12 cells. Taken together, these findings indicate that SIRT1 activation may be a useful strategy for treating muscular dystrophies.

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