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Rationale: Autophagy, a bulk degradation process of cytosolic proteins and organelles, is protective during nutrient starvation in cardiomyocytes (CMs). However, the underlying signaling mechanism mediating autophagy is not well understood. Objective:We investigated the role of FoxOs and its posttranslational modification in mediating starvationinduced autophagy. Methods and Results:Glucose deprivation (GD) increased autophagic flux in cultured CMs, as evidenced by increased mRFP-GFP-LC3 puncta and decreases in p62, which was accompanied by upregulation of Sirt1 and FoxO1. Overexpression of either Sirt1 or FoxO1 was sufficient for inducing autophagic flux, whereas both Sirt1 and FoxO1 were required for GD-induced autophagy. GD increased deacetylation of FoxO1, and Sirt1 was required for GD-induced deacetylation of FoxO1. Overexpression of FoxO1(3A/LXXAA), which cannot interact with Sirt1, or p300, a histone acetylase, increased acetylation of FoxO1 and inhibited GD-induced autophagy. FoxO1 increased expression of Rab7, a small GTP-binding protein that mediates late autophagosome-lysosome fusion, which was both necessary and sufficient for mediating FoxO1-induced increases in autophagic flux. Although cardiac function was maintained in control mice after 48 hours of food starvation, it was significantly deteriorated in mice with cardiac-specific overexpression of FoxO1(3A/LXXAA), those with cardiac-specific homozygous deletion of FoxO1 (c-FoxO1 Key Words: autophagy Ⅲ starvation Ⅲ FoxO Ⅲ Sirt1 Ⅲ Rab7 Ⅲ deacetylation M acroautophagy (termed hereafter as autophagy) is a dynamic process of intracellular bulk degradation in which cytosolic proteins and organelles are sequestered into double-membrane vesicles called autophagosomes to be fused with lysosomes for degradation. 1 In the heart, autophagy maintains protein quality control, adapts to nutrient and oxygen deprivation during myocardial ischemia, and mediates cell death during reperfusion injury. 2,3 Autophagy during nutrient deprivation is an adaptive mechanism that allows the cells to survive by degrading the intracellular protein and lipid cargo and recycling the amino and fatty acids to generate ATP. 2 The nutrient status has a profound effect on cardiac contractility, and activation of autophagy during starvation is protective for the heart. The intracellular signaling mechanism by which nutrient starvation activates autophagy in cardiomyocytes (CMs) is not well understood, however.The forkhead box, class O (FoxO) family of transcription factors are present as 4 distinct isoforms (FoxO1, FoxO3, FoxO4, and FoxO6) in mammals. FoxO proteins play an important role in several intracellular functions, such as metabolism, stress resistance, longevity, tumor suppression, and cell size regulation. 4 The key to the myriad functions of FoxO proteins lies in the complex posttranslational modifications they undergo. They are phosphorylated in response to insulin and growth factors, dephosphorylated by protein phosphatases, ubiquitinated in response to oxidative stre...

Background-Silent information regulator 1 (Sirt1), a class III histone deacetylase, retards aging and protects the heart from oxidative stress. We here examined whether Sirt1 is protective against myocardial ischemia/reperfusion (I/R). Methods and Results-Protein and mRNA expression of Sirt1 is significantly reduced by I/R. Cardiac-specific Sirt1 Ϫ/Ϫ mice exhibited a significant increase (44Ϯ5% versus 15Ϯ5%; Pϭ0.01) in the size of myocardial infarction/area at risk. In transgenic mice with cardiac-specific overexpression of Sirt1, both myocardial infarction/area at risk (15Ϯ4% versus 36Ϯ8%; Pϭ0.004) and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (4Ϯ3% versus 10Ϯ1%; PϽ0.003) were significantly reduced compared with nontransgenic mice. In Langendorff-perfused hearts, the functional recovery during reperfusion was significantly greater in transgenic mice with cardiac-specific overexpression of Sirt1 than in nontransgenic mice. Sirt1 positively regulates expression of prosurvival molecules, including manganese superoxide dismutase, thioredoxin-1, and Bcl-xL, whereas it negatively regulates the proapoptotic molecules Bax and cleaved caspase-3. The level of oxidative stress after I/R, as evaluated by anti-8-hydroxydeoxyguanosine staining, was negatively regulated by Sirt1. Sirt1 stimulates the transcriptional activity of FoxO1, which in turn plays an essential role in mediating Sirt1-induced upregulation of manganese superoxide dismutase and suppression of oxidative stress in cardiac myocytes. Sirt1 plays an important role in mediating I/R-induced increases in the nuclear localization of FoxO1 in vivo. Conclusions-These results suggest that Sirt1 protects the heart from I/R injury through upregulation of antioxidants and downregulation of proapoptotic molecules through activation of FoxO and decreases in oxidative stress. (Circulation. 2010;122:2170-2182.)Key Words: cardioprotection Ⅲ ischemia Ⅲ oxidative stress Ⅲ reperfusion injury S ilent information regulator 1 (Sirt1) is a member of the sirtuin family of class III histone deacetylases. 1 The class III histone deacetylases are distinguished from histone deacetylases in the other classes by their requirement of NAD ϩ for their enzyme activity. 2 Sirt1 is involved in gene silencing, differentiation, cell survival, metabolism, and longevity. 1 Sirt1 activity extends the lifespan of lower organisms, including yeast, Caenorhabditis elegans, and flies. 3,4 In addition, resveratrol, which stimulates Sirt1, extends the lifespan of mice fed a high-fat diet, suggesting that Sirt1 may affect aging and/or lifespan in mammals. 5 The beneficial effects of caloric restriction may be dependent on Sirt1. 6 -8 Conversely, Sirt1 knockout mice exhibit developmental abnormalities, including septal and valvular heart defects. 9,10 Sirt1 regulates the function of transcription factors and cofactors, including MyoD, Ku, p53, PGC1, and the FoxO family of transcription factors, 11-19 through deacetylation. Clinical Perspective on p 2182Activation of mole...

Autophagy is a bulk degradation process in which cytosolic proteins and organelles are degraded through lysosomes. To evaluate autophagic flux in cardiac myocytes, we generated adenovirus and cardiac-specific transgenic mice harboring tandem fluorescent mRFP-GFP-LC3. Starvation significantly increased the number of mRFP-GFP-LC3 dots representing both autophagosomes and autolysosomes per cell, suggesting that autophagic flux is increased in cardiac myocytes. H 2 O 2 significantly increased autophagic flux, which was attenuated in the presence of N-2-mercaptopropionyl glycine (MPG), an antioxidant, suggesting that oxidative stress stimulates autophagy in cardiac myocytes. Myocardial ischemia=reperfusion (I=R) increased both autophagosomes and autolysosomes, thereby increasing autophagic flux. Treatment with MPG attenuated I=R-induced increases in oxidative stress, autophagic flux, and Beclin-1 expression, accompanied by a decrease in the size of myocardial infarction (MI)=area at risk (AAR), suggesting that oxidative stress plays an important role in mediating autophagy and myocardial injury during I=R. MI=AAR after I=R was significantly reduced in beclin1 +=À mice, whereas beclin1 +=À mice treated with MPG exhibited no additional reduction in the size of MI=AAR after I=R. These results suggest that oxidative stress plays an important role in mediating autophagy during I=R, and that activation of autophagy through oxidative stress mediates myocardial injury in response to I=R in the mouse heart.

Rationale: NAD؉ acts not only as a cofactor for cellular respiration but also as a substrate for NAD ؉ -dependent enzymes, such as Sirt1. The cellular NAD ؉ synthesis is regulated by both the de novo and the salvage pathways. Nicotinamide phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the salvage pathway. Objective: Here we investigated the role of Nampt in mediating NAD ؉ synthesis in cardiac myocytes and the function of Nampt in the heart in vivo. Methods and Results: Expression of Nampt in the heart was significantly decreased by ischemia, ischemia/ reperfusion and pressure overload. Upregulation of Nampt significantly increased NAD ؉ and ATP concentrations, whereas downregulation of Nampt significantly decreased them. Downregulation of Nampt increased caspase 3 cleavage, cytochrome c release, and TUNEL-positive cells, which were inhibited in the presence of Bcl-xL, but did not increase hairpin 2-positive cells, suggesting that endogenous Nampt negatively regulates apoptosis but not necrosis. Downregulation of Nampt also impaired autophagic flux, suggesting that endogenous Nampt positively regulates autophagy. Cardiac-specific overexpression of Nampt in transgenic mice increased NAD ؉ content in the heart, prevented downregulation of Nampt, and reduced the size of myocardial infarction and apoptosis in response to prolonged ischemia and ischemia/reperfusion. Conclusions: Nampt critically regulates NAD ؉ and ATP contents, thereby playing an essential role in mediating cell survival by inhibiting apoptosis and stimulating autophagic flux in cardiac myocytes. Preventing downregulation of Nampt inhibits myocardial injury in response to myocardial ischemia and reperfusion. These results suggest that Nampt is an essential gatekeeper of energy status and survival in cardiac myocytes. Because of its involvement in the mitochondrial TCA cycle and the electron transport chain, NAD ϩ acts as a key cofactor for energy production. NAD ϩ also serves as the substrate for various enzymes, including the nuclear enzyme poly(ADPribose) polymerase (PARP)-1, 1 and the class III histone deacetylases, ie, the sirtuin family. 2 Because the sirtuin family plays an essential role in mediating lifespan extension, stress resistance and regulation of metabolism, 3 NAD ϩ may control the level of stress resistance in cells partly through regulation of sirtuins. 4 NAD ϩ can be freshly synthesized from amino acids, including tryptophan or aspartic acid, via the de novo pathway 5 or taken up efficiently from the extracellular space. 6 Importantly, NAD ϩ can also be resynthesized from NAD ϩ metabolites through the salvage pathway. 5 In yeast, increased expression of pyrazinamidase/nicotinamidase 1, a nicotinamidase converting nicotinamide to nicotinic acid, is both necessary and sufficient for lifespan extension induced by calorie restriction and low-intensity stress, such as osmotic stress. 7 Nicotinamide phosphoribosyltransferase (Nampt) is a ratelimiting enzyme in the mammalian NAD ϩ salvage pathway, and has been prop...

Rationale Myocardial function is enhanced by adoptive transfer of human cardiac progenitor cells (hCPCs) into a pathologically challenged heart. However, advanced age, comorbidities, and myocardial injury in patients with heart failure constrain the proliferation, survival, and regenerative capacity of hCPCs. Rejuvenation of senescent hCPCs will improve the outcome of regenerative therapy for a substantial patient population possessing functionally impaired stem cells. Objective Reverse phenotypic and functional senescence of hCPCs by ex vivo modification with Pim-1. Methods and Results C-kit–positive hCPCs were isolated from heart biopsy samples of patients undergoing left ventricular assist device implantation. Growth kinetics, telomere lengths, and expression of cell cycle regulators showed significant variation between hCPC isolated from multiple patients. Telomere length was significantly decreased in hCPC with slow-growth kinetics concomitant with decreased proliferation and upregulation of senescent markers compared with hCPC with fast-growth kinetics. Desirable youthful characteristics were conferred on hCPCs by genetic modification using Pim-1 kinase, including increases in proliferation, telomere length, survival, and decreased expression of senescence markers. Conclusions Senescence characteristics of hCPCs are ameliorated by Pim-1 kinase resulting in rejuvenation of phenotypic and functional properties. hCPCs show improved cellular properties resulting from Pim-1 modification, but benefits were more pronounced in hCPC with slow-growth kinetics relative to hCPC with fast-growth kinetics. With the majority of patients with heart failure presenting advanced age, infirmity, and impaired regenerative capacity, the use of Pim-1 modification should be incorporated into cell-based therapeutic approaches to broaden inclusion criteria and address limitations associated with the senescent phenotype of aged hCPC.

Autophagy is an intracellular bulk degradation process whereby cytoplasmic proteins and organelles are degraded and recycled through lysosomes. In the heart, autophagy plays a homeostatic role at basal levels, and the absence of autophagy causes cardiac dysfunction and the development of cardiomyopathy. Autophagy is induced during myocardial ischemia and further enhanced by reperfusion. Although induction of autophagy during the ischemic phase is protective, further enhancement of autophagy during the reperfusion phase may induce cell death and appears to be detrimental. In this review we discuss the functional significance of autophagy and the underlying signaling mechanism in the heart during ischemia/reperfusion.

Autophagy is a catabolic process which degrades long-lived proteins and damaged organelles through sequestration into double membrane structures termed autophagosomes and fusion with lysosomes. Autophagy is active in the heart at baseline and further stimulated under stress conditions, including starvation, ischemia/reperfusion and heart failure. Autophagy plays an adaptive role in the heart at baseline, thereby maintaining cardiac structure and function and inhibiting age-related cardiac abnormalities. Autophagy is activated by ischemia and nutrient starvation in the heart through Sirt1-FoxO and AMPK-dependent mechanisms, respectively. Activation of autophagy during ischemia is essential for cell survival and maintenance of cardiac function. Autophagy is strongly activated in the heart during reperfusion after ischemia. Activation of autophagy during reperfusion could be either protective or detrimental, depending upon the experimental model. However, strong induction of autophagy accompanied by robust upregulation of Beclin1 could cause autophagic cell death, thereby being detrimental. This review provides an overview regarding both protective and detrimental functions of autophagy in the heart and discusses possible applications of current knowledge to the treatment of heart disease.