Nirmala Hariharan

Deacetylation of FoxO by Sirt1 Plays an Essential Role in Mediating Starvation-Induced Autophagy in Cardiac Myocytes

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 starvation-induced 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−/−), and beclin1+/− mice, in which autophagy is significantly inhibited. Conclusions: These results suggest that Sirt1-mediated deacetylation of FoxO1 and upregulation of Rab7 play an important role in mediating starvation-induced increases in autophagic flux, which in turn plays an essential role in maintaining left ventricular function during starvation.

Silent Information Regulator 1 Protects the Heart From Ischemia/Reperfusion

Background 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).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−/− mice exhibited a significant increase (44±5% versus 15±5%; P=0.01) in the size of myocardial infarction/area at risk. In transgenic mice with cardiac-specific overexpression of Sirt1, both myocardial infarction/area at risk (15±4% versus 36±8%; P=0.004) and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (4±3% versus 10±1%; P<0.003) 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.

Oxidative Stress Stimulates Autophagic Flux During Ischemia/Reperfusion

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(+/-) mice, whereas beclin1(+/-) 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.

Nicotinamide Phosphoribosyltransferase Regulates Cell Survival Through NAD+ Synthesis in Cardiac Myocytes

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.

Molecular mechanisms and physiological significance of autophagy during myocardial ischemia and reperfusion

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.

Is Autophagy in Response to Ischemia and Reperfusion Protective or Detrimental for the Heart

Autophagy is a catabolic process that degrades long-lived proteins and damaged organelles by sequestering them into double membrane structures termed “autophagosomes” and fusing them with lysosomes. Autophagy is active in the heart at baseline and further stimulated under stress conditions including starvation, ischemia/reperfusion, and heart failure. It 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 adenosine monophosphate (AMP)-activated protein kinase (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 on the experimental model. However, strong induction of autophagy accompanied by robust upregulation of Beclin1 could cause autophagic cell death, thereby proving to be 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.

Novel methods for measuring cardiac autophagy in vivo.

Autophagy, a highly conserved cellular mechanism wherein various cellular components are broken down and recycled through lysosomes, occurs constitutively in the heart and may serve as a cardioprotective mechanism in some situations. It has been implicated in the development of heart failure and is up-regulated following ischemia-reperfusion injury. Autophagic flux, a measure of autophagic vesicle formation and clearance, is an important measurement in evaluating the efficacy of the pathway, however, tools to measure flux in vivo have been limited. Here, we describe the use of monodansylcadaverine (MDC) and the lysosomotropic drug chloroquine to measure autophagic flux in in vivo model systems, specifically focusing on its use in the myocardium. This method allows determination of flux as a more precise measure of autophagic activity in vivo much in the same way that Bafilomycin A(1) is used to measure flux in cell culture. MDC injected 1 h before sacrifice, colocalizes with mCherry-LC3 puncta, validating its use as a marker of autophagosomes. This chapter provides a method to measure autophagic flux in vivo in both transgenic and nontransgenic animals, using MDC and chloroquine, and in addition describes the mCherry-LC3 mouse and the advantages of this animal model in the study of cardiac autophagy. Additionally, we review several methods for inducing autophagy in the myocardium under pathological conditions such as myocardial infarction, ischemia/ reperfusion, pressure overloading, and nutrient starvation.

Autophagy plays an essential role in mediating regression of hypertrophy during unloading of the heart.

Autophagy is a bulk degradation mechanism for cytosolic proteins and organelles. The heart undergoes hypertrophy in response to mechanical load but hypertrophy can regress upon unloading. We hypothesize that autophagy plays an important role in mediating regression of cardiac hypertrophy during unloading. Mice were subjected to transverse aortic constriction (TAC) for 1 week, after which the constriction was removed (DeTAC). Regression of cardiac hypertrophy was observed after DeTAC, as indicated by reduction of LVW/BW and cardiomyocyte cross-sectional area. Indicators of autophagy, including LC3-II expression, p62 degradation and GFP-LC3 dots/cell, were significantly increased after DeTAC, suggesting that autophagy is induced. Stimulation of autophagy during DeTAC was accompanied by upregulation of FoxO1. Upregulation of FoxO1 and autophagy was also observed in vitro when cultured cardiomyocytes were subjected to mechanical stretch followed by incubation without stretch (de-stretch). Transgenic mice with cardiac-specific overexpression of FoxO1 exhibited smaller hearts and upregulation of autophagy. Overexpression of FoxO1 in cultured cardiomyocytes significantly reduced cell size, an effect which was attenuated when autophagy was inhibited. To further examine the role of autophagy and FoxO1 in mediating the regression of cardiac hypertrophy, beclin1+/− mice and cultured cardiomyocytes transduced with adenoviruses harboring shRNA-beclin1 or shRNA-FoxO1 were subjected to TAC/stretch followed by DeTAC/de-stretch. Regression of cardiac hypertrophy achieved after DeTAC/de-stretch was significantly attenuated when autophagy was suppressed through downregulation of beclin1 or FoxO1. These results suggest that autophagy and FoxO1 play an essential role in mediating regression of cardiac hypertrophy during mechanical unloading.

Nicotinamide phosphoribosyltransferase regulates cell survival through autophagy in cardiomyocytes.

Nicotinamide adenine dinucleotide (NAD+) acts as a transfer molecule for electrons, thereby acting as a key cofactor for energy production. NAD+ also serves as a substrate for cellular enzymes, including poly (ADP-ribose) polymerase (PARP)-1 and Sirt1. Activation of PARP-1 by DNA damage depletes the cellular pool of NAD+, leading to necrotic cell death. NAD+ in the nucleus enhances the activity of Sirt1, thereby modulating transcription. NAD+ is either synthesized de novo from amino acids, namely tryptophan and aspartic acid, or resynthesized from NAD+ metabolites, such as nicotinamide (NAM), through the salvage pathway. NAM phosphoribosyltransferase (Nampt) is a rate-limiting enzyme in the NAD+ salvage pathway. We have recently demonstrated that Nampt is an important regulator of NAD+ and autophagy in cardiomyocytes. Here we discuss the role of Nampt in regulating autophagy and potential mechanisms by which NAD+ regulates autophagy in the heart.

Sirt1 Protects the Heart from Ischemia/Reperfusion

Background 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).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−/− mice exhibited a significant increase (44±5% versus 15±5%; P=0.01) in the size of myocardial infarction/area at risk. In transgenic mice with cardiac-specific overexpression of Sirt1, both myocardial infarction/area at risk (15±4% versus 36±8%; P=0.004) and terminal deoxynucleotidyl transferase dUTP nick end labeling-positive nuclei (4±3% versus 10±1%; P<0.003) 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.