Sirtuin 1 Promotes Hyperoxia-Induced Lung Epithelial Cell Death Independent of NF-E2-Related Factor 2 Activation.

Lung epithelial cell damage accompanied by death is a cardinal feature of toxicant- and prooxidant-induced acute lung injury. The transcription factor nuclear factor (erythroid-derived 2)-like 2 (NEF2L2 or NRF2) activates several antioxidant enzymes (AOEs) and prosurvival genes in response to oxidant stress, and its deficiency enhances susceptibility to hyperoxic lung injury and other oxidant-induced lung pathologies. Sirtuin 1 (SIRT1) regulates cell growth and survival in response to both physiological and pathological stresses by selectively deacetylating multiple proteins required for chromatin remodeling and transcription; therefore, we sought to examine potential SIRT1-NRF2 cross-talk in the regulation of AOE expression during hyperoxia-induced lung epithelial cell death. Unexpectedly, pharmacological inhibition or small interfering RNA-mediated depletion of SIRT1 caused a reduction in cell death, accompanied by reduced levels of NRF2-dependent AOE expression in chronic hyperoxia. NRF2 acetylation was markedly and transiently higher in cells exposed to acute (6 h) hyperoxia. Sirtinol blocked this acute effect, but NRF2 acetylation was low or undetectable in cells exposed to chronic hyperoxia (24-36 h) both with and without sirtinol. SIRT1 activation by resveratrol augmented hyperoxia-induced death in cells with NRF2 deficiency. SIRT1 inhibition or depletion led to a reduced activation of the cell-death executioner caspase 3, whereas caspase inhibition prevented death. Consistent with these results, sirtinol attenuated hyperoxia-induced lung alveolar permeability and toxicity in vivo. Collectively, these results reveal that, in chronic hyperoxia, SIRT1 promotes hyperoxia-induced lung epithelial cell damage and death by altering pro- and antiapoptotic balance, not by dampening optimal NRF2-dependent AOE expression.

NADPH oxidase-derived H(2)O(2) contributes to angiotensin II-induced aldosterone synthesis in human and rat adrenal cortical cells.

BACKGROUND: The Renin-Angiotensin-Aldosterone-System plays a pivotal role in hypertension. Angiotensin II (Ang II) is a major regulator of aldosterone synthesis and secretion, and it is known to facilitate reactive oxygen species (ROS) generation in many cell types. AIMS: Here, we assessed the role of ROS signaling in Ang II-induced aldosterone synthesis by focusing on the regulation of aldosterone synthase (CYP11B2), a cytochrome P450 oxidase that catalyzes the final step in aldosterone biosynthetic pathway. RESULTS: Ang II increased CYP11B2 activity, mRNA and protein with a concomitant elevation of 6-Carboxy- 2',7'-dichlorodihydrofluorescein diacetate fluorescence, malondialdehyde and protein carbonyl levels (indices of ROS), NADPH oxidase (Nox) activity, and H(2)O(2) levels in human and rat adrenal cortical cells. The expression of nuclear receptor related 1 protein, a transcription factor known to regulate CYP11B2 expression, was also augmented by Ang II. These Ang II-evoked effects were either abolished or attenuated by pretreatment of cells with either Ang II type I receptor (AT(1)R) antagonist, or antioxidants or Nox inhibitor or siRNA silencing of Nox1, 2 and 4, or inhibitors of phospholipase C and protein kinase C. Exogenous H(2)O(2) mimicked the facilitatory effects of Ang II on CYP11B2 activity, mRNA, and protein expression, and these changes were significantly reduced by PEG-catalase. INNOVATION: ROS, particularly H(2)O(2), is identified as a key regulator of aldosterone production. CONCLUSION: Our results suggest that Ang II facilitates CYP11B2 activity and the ensuing aldosterone production via activation of AT(1)R-Nox-H(2)O(2) signaling pathway.

Exogenous NAD blocks cardiac hypertrophic response via activation of the SIRT3-LKB1-AMP-activated kinase pathway.

Since the discovery of NAD-dependent deacetylases, sirtuins, it has been recognized that maintaining intracellular levels of NAD is crucial for the management of stress response of cells. Here we show that agonist-induced cardiac hypertrophy is associated with loss of intracellular levels of NAD, but not exercise-induced physiologic hypertrophy. Exogenous addition of NAD was capable of maintaining intracellular levels of NAD and blocking the agonist-induced cardiac hypertrophic response in vitro as well as in vivo. NAD treatment blocked the activation of pro-hypertrophic Akt1 signaling, and augmented the activity of anti-hypertrophic LKB1-AMPK signaling in the heart, which prevented subsequent induction of mTOR-mediated protein synthesis. By using gene knock-out and transgenic mouse models of SIRT3 and SIRT1, we showed that the anti-hypertrophic effects of exogenous NAD are mediated through activation of SIRT3, but not SIRT1. SIRT3 deacetylates and activates LKB1, thus augmenting the activity of the LKB1-AMPK pathway. These results reveal a novel role of NAD as an inhibitor of cardiac hypertrophic signaling, and suggest that prevention of NAD depletion may be critical in the treatment of cardiac hypertrophy and heart failure.

Sirt3 blocks the cardiac hypertrophic response by augmenting Foxo3a-dependent antioxidant defense mechanisms in mice.

Sirtuin 3 (SIRT3) is a member of the sirtuin family of proteins that promote longevity in many organisms. Increased expression of SIRT3 has been linked to an extended life span in humans. Here, we have shown that Sirt3 protects the mouse heart by blocking the cardiac hypertrophic response. Although Sirt3-deficient mice appeared to have normal activity, they showed signs of cardiac hypertrophy and interstitial fibrosis at 8 weeks of age. Application of hypertrophic stimuli to these mice produced a severe cardiac hypertrophic response, whereas Sirt3-expressing Tg mice were protected from similar stimuli. In primary cultures of cardiomyocytes, Sirt3 blocked cardiac hypertrophy by activating the forkhead box O3a-dependent (Foxo3a-dependent), antioxidant-encoding genes manganese superoxide dismutase (MnSOD) and catalase (Cat), thereby decreasing cellular levels of ROS. Reduced ROS levels suppressed Ras activation and downstream signaling through the MAPK/ERK and PI3K/Akt pathways. This resulted in repressed activity of transcription factors, specifically GATA4 and NFAT, and translation factors, specifically eukaryotic initiation factor 4E (elf4E) and S6 ribosomal protein (S6P), which are involved in the development of cardiac hypertrophy. These results demonstrate that SIRT3 is an endogenous negative regulator of cardiac hypertrophy, which protects hearts by suppressing cellular levels of ROS.

SIRT1 promotes cell survival under stress by deacetylation-dependent deactivation of poly(ADP-ribose) polymerase 1.

Poly(ADP-ribose) polymerase 1 (PARP1) and SIRT1 deacetylase are two NAD-dependent enzymes which play major roles in the decision of a cell to live or to die in a stress situation. Because of the dependence of both enzymes on NAD, cross talk between them has been suggested. Here, we show that PARP1 is acetylated after stress of cardiomyocytes, resulting in the activation of PARP1, which is independent of DNA damage. SIRT1 physically binds to and deacetylates PARP1. Increased acetylation of PARP1 was also detected in hearts of SIRT1(-/-) mice, compared to that detected in the hearts of SIRT1(+/+) mice, confirming a role of SIRT1 in regulating the PARP1 acetylation in vivo. SIRT1-dependent deacetylation blocks PARP1 activity, and it protects cells from PARP1-mediated cell death. We also show that SIRT1 negatively regulates the activity of the PARP1 gene promoter, thus suggesting that the deacetylase controls the PARP1 activity at the transcriptional level as well. These data demonstrate that the activity of PARP1 is under the control of SIRT1, which is necessary for survival of cells under stress conditions.

SIRT3 is a stress-responsive deacetylase in cardiomyocytes that protects cells from stress-mediated cell death by deacetylation of Ku70.

There are seven SIRT isoforms in mammals, with diverse biological functions including gene regulation, metabolism, and apoptosis. Among them, SIRT3 is the only sirtuin whose increased expression has been shown to correlate with an extended life span in humans. In this study, we examined the role of SIRT3 in murine cardiomyocytes. We found that SIRT3 is a stress-responsive deacetylase and that its increased expression protects myocytes from genotoxic and oxidative stress-mediated cell death. We show that, like human SIRT3, mouse SIRT3 is expressed in two forms, a approximately 44-kDa long form and a approximately 28-kDa short form. Whereas the long form is localized in the mitochondria, nucleus, and cytoplasm, the short form is localized exclusively in the mitochondria of cardiomyocytes. During stress, SIRT3 levels are increased not only in mitochondria but also in the nuclei of cardiomyocytes. We also identified Ku70 as a new target of SIRT3. SIRT3 physically binds to Ku70 and deacetylates it, and this promotes interaction of Ku70 with the proapoptotic protein Bax. Thus, under stress conditions, increased expression of SIRT3 protects cardiomyocytes, in part by hindering the translocation of Bax to mitochondria. These studies underscore an essential role of SIRT3 in the survival of cardiomyocytes in stress situations.

Activation of SIRT1, a class III histone deacetylase, contributes to fructose feeding-mediated induction of the alpha-myosin heavy chain expression.

Fructose feeding has been shown to induce the cardiac alpha-myosin heavy chain (MHC) expression and protect the heart from ischemia- and reperfusion-mediated cell injury. This study was designed to investigate the mechanism involved in the effect of this sugar on MHC gene expression and cardiac protection. Adult mice were fed with a 6-propyl-2-thiouracil (PTU) diet or PTU combined with a fructose-rich diet. PTU treatment made animals hypothyroid and that resulted in total replacement of cardiac alpha-MHC with the beta-MHC isoform. Addition of fructose in the PTU diet led to reexpression of the alpha-MHC isoform to a significant level. Similar induction of alpha-MHC expression was also seen when PTU diet was combined with resveratrol, an agonist of sirtuin (SIRT) 1 deacetylase. Analysis of heart lysate of these animals indicated that fructose feeding augmented the NAD-to-NADH ratio and the cardiac SIRT1 levels, thus suggesting a role of SIRT1 in fructose-mediated activation of alpha-MHC isoform. To analyze a direct effect of SIRT1 on MHC isoform expression, we generated transgenic mice expressing SIRT1 in the heart. Treatment of these transgenic mice with PTU diet did not lead to disappearance of alpha-MHC, as it did in the nontransgenic animals. SIRT1 overexpression also activated the alpha-MHC gene promoter in transient transfection assays, thus confirming a role of SIRT1 in the induction of alpha-MHC expression. Fructose feeding also attenuated the MHC isoform shift and blocked the cardiac hypertrophy response associated with pressure overload, which was again associated with the induction of cardiac SIRT1 levels. These results demonstrate that fructose feeding protects the heart by induction of the SIRT1 deacetylase and highlight its role in the induction of alpha-MHC gene expression.

Poly(ADP-ribose) polymerase-1-deficient mice are protected from angiotensin II-induced cardiac hypertrophy.

Poly(ADP-ribose) polymerase-1 (PARP), a chromatin-bound enzyme, is activated by cell oxidative stress. Because oxidative stress is also considered a main component of angiotensin II-mediated cell signaling, it was postulated that PARP could be a downstream target of angiotensin II-induced signaling leading to cardiac hypertrophy. To determine a role of PARP in angiotensin II-induced hypertrophy, we infused angiotensin II into wild-type (PARP(+/+)) and PARP-deficient mice. Angiotensin II infusion significantly increased heart weight-to-tibia length ratio, myocyte cross-sectional area, and interstitial fibrosis in PARP(+/+) but not in PARP(-/-) mice. To confirm these results, we analyzed the effect of angiotensin II in primary cultures of cardiomyocytes. When compared with PARP(-/-) cardiomyocytes, angiotensin II (1 microM) treatment significantly increased protein synthesis in PARP(+/+) myocytes, as measured by (3)H-leucine incorporation into total cell protein. Angiotensin II-mediated hypertrophy of myocytes was accompanied with increased poly-ADP-ribosylation of nuclear proteins and depletion of cellular NAD content. When cells were treated with cell death-inducing doses of angiotensin II (10-20 microM), robust myocyte cell death was observed in PARP(+/+) but not in PARP(-/-) myocytes. This type of cell death was blocked by repletion of cellular NAD levels as well as by activation of the longevity factor Sir2alpha deacetylase, indicating that PARP induction and subsequent depletion of NAD levels are the sequence of events causing angiotensin II-mediated cardiomyocyte cell death. In conclusion, these results demonstrate that PARP is a nuclear integrator of angiotensin II-mediated cell signaling contributing to cardiac hypertrophy and suggest that this could be a novel therapeutic target for the management of heart failure.