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 (ADPribose) 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 aging and stress.

The prevalence of heart diseases, such as coronary artery disease and congestive heart failure, increases with age. Optimal therapeutic interventions that antagonize aging may reduce the occurrence and mortality of adult heart diseases. We discuss here how molecular mechanisms mediating life span extension affect aging of the heart and its resistance to pathological insults. In particular, we review our recent findings obtained from transgenic mice with cardiac-specific overexpression of Sirt1, which demonstrated delayed aging and protection against oxidative stress in the heart. We propose that activation of known longevity mechanisms in the heart may represent a novel cardioprotection strategy against aging and certain types of cardiac stress, such as oxidative stress.

Sirt1 regulates aging and resistance to oxidative stress in the heart.

Silent information regulator (Sir)2, a class III histone deacetylase, mediates lifespan extension in model organisms and prevents apoptosis in mammalian cells. However, beneficial functions of Sir2 remain to be shown in mammals in vivo at the organ level, such as in the heart. We addressed this issue by using transgenic mice with heart-specific overexpression of Sirt1, a mammalian homolog of Sir2. Sirt1 was significantly upregulated (4- to 8-fold) in response to pressure overload and oxidative stress in nontransgenic adult mouse hearts. Low (2.5-fold) to moderate (7.5-fold) overexpression of Sirt1 in transgenic mouse hearts attenuated age-dependent increases in cardiac hypertrophy, apoptosis/fibrosis, cardiac dysfunction, and expression of senescence markers. In contrast, a high level (12.5-fold) of Sirt1 increased apoptosis and hypertrophy and decreased cardiac function, thereby stimulating the development of cardiomyopathy. Moderate overexpression of Sirt1 protected the heart from oxidative stress induced by paraquat, with increased expression of antioxidants, such as catalase, through forkhead box O (FoxO)-dependent mechanisms, whereas high levels of Sirt1 increased oxidative stress in the heart at baseline. Thus, mild to moderate expression of Sirt1 retards aging of the heart, whereas a high dose of Sirt1 induces cardiomyopathy. Furthermore, although high levels of Sirt1 increase oxidative stress, moderate expression of Sirt1 induces resistance to oxidative stress and apoptosis. These results suggest that Sirt1 could retard aging and confer stress resistance to the heart in vivo, but these beneficial effects can be observed only at low to moderate doses (up to 7.5-fold) of Sirt1.

Histone H2A.z is essential for cardiac myocyte hypertrophy but opposed by silent information regulator 2alpha.

In this study we have shown that the histone variant H2A.z is up-regulated during cardiac hypertrophy. Upon its knock-down with RNA interference, hypertrophy and the underlying increase in growth-related genes, protein synthesis, and cell size were down-regulated. During attempts to understand the mode of regulation of H2A.z, we found that overexpression of silent information regulator 2alpha (Sir2alpha) specifically induced down-regulation of H2A.z via NAD-dependent activity. This effect was reversed by the proteasome inhibitor epoxomicin, suggesting a Sir2alpha-mediated ubiquitin/proteasome-dependent mechanism for degradation of H2A.z. An increase in Sir2alpha also resulted in a dose-dependent reduction of the response to hypertrophic stimuli, whereas its inhibition resulted in enhanced hypertrophy and apoptosis. We have shown that Sir2alpha directly deacetylates H2A.z. Mutagenesis proved that lysines 4, 7, 11, and 13 do not play a role in the stability of H2A.z, whereas Lys-15 was indispensable. Meanwhile, Lys-115 and conserved, ubiquitinatable Lys-121 are critical for Sir2alpha-mediated degradation. Fusion of the C terminus of H2A.z (amino acids 115-127) to H2A.x or green fluorescence protein conferred Sir2alpha-inducible degradation to the former protein only. Because H2A.x and H2A.z have conserved N-tails, this implied that both the C and N termini are critical for mediating the effect of Sir2alpha. In short, the results suggest that H2A.z is required for cardiac hypertrophy, where its stability and the extent of cell growth and apoptosis are moderated by Sir2alpha. We also propose that Sir2alpha is involved in deacetylation of H2A.z, which results in ubiquitination of Lys-115 and Lys-121 and its degradation via a ubiquitin/proteasome-dependent pathway.

Caspase-3 mediated cleavage of MEKK1 promotes p53 transcriptional activity.

Myocardial ischemia/reperfusion (IR) induces myocyte apoptosis, and the pro-apoptotic/tumor suppressor protein p53 may contribute to this process. However, the signaling mechanism by which IR induces p53 activation remains largely unknown. Here, we show that MEKK1 undergoes proteolytic cleavage in a caspase-3 dependent manner in both in vivo and in vitro models of ischemic injury. Overexpression studies both in vivo and in vitro indicated that the caspase-3 mediated cleavage of MEKK1 promotes phosphorylation and transcriptional activity of p53. In addition, caspase-3 inhibited the ability of the wild-type full-length form of MEKK1 to activate ATF2, suggesting that caspase-3, by way of proteolytic cleavage, abrogates the ability of MEKK1 to signal JNK. We propose that IR induces caspase-3 mediated proteolytic cleavage of MEKK1 and promotes p53 transcriptional activity via JNK-independent mechanisms, which in turn may contribute to pathological insults associated with IR injury, such as myocyte apoptosis.

Silent information regulator 2alpha, a longevity factor and class III histone deacetylase, is an essential endogenous apoptosis inhibitor in cardiac myocytes.

Yeast silent information regulator 2 (Sir2), a nicotinamide adenine dinucleotide-dependent histone deacetylase (HDAC) and founding member of the HDAC class III family, functions in a wide array of cellular processes, including gene silencing, longevity, and DNA damage repair. We examined whether or not the mammalian ortholog Sir2 affects growth and death of cardiac myocytes. Cardiac myocytes express Sir2alpha predominantly in the nucleus. Neonatal rat cardiac myocytes were treated with 20 mmol/L nicotinamide (NAM), a Sir2 inhibitor, or 50 nmol/L Trichostatin A (TSA), a class I and II HDAC inhibitor. NAM induced a significant increase in nuclear fragmentation (2.2-fold) and cleaved caspase-3, as did sirtinol, a specific Sir2 inhibitor, and expression of dominant-negative Sir2alpha. TSA also modestly increased cell death (1.5-fold) but without accompanying caspase-3 activation. Although TSA induced a 1.5-fold increase in cardiac myocyte size and protein content, NAM reduced both. In addition, NAM caused acetylation and increases in the transcriptional activity of p53, whereas TSA did not. NAM-induced cardiac myocyte apoptosis was inhibited in the presence of dominant-negative p53, suggesting that Sir2alpha inhibition causes apoptosis through p53. Overexpression of Sir2alpha protected cardiac myocytes from apoptosis in response to serum starvation and significantly increased the size of cardiac myocytes. Furthermore, Sir2 expression was increased significantly in hearts from dogs with heart failure induced by rapid pacing superimposed on stable, severe hypertrophy. These results suggest that endogenous Sir2alpha plays an essential role in mediating cell survival, whereas Sir2alpha overexpression protects myocytes from apoptosis and causes modest hypertrophy. In contrast, inhibition of endogenous class I and II HDACs primarily causes cardiac myocyte hypertrophy and also induces modest cell death. An increase in Sir2 expression during heart failure suggests that Sir2 may play a cardioprotective role in pathologic hearts in vivo.