Published online before print October 14, 2004, doi: 10.1161/01.RES.0000147557.75257.ff
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© 2004 American Heart Association, Inc.
Molecular Medicine |
Silent Information Regulator 2
, a Longevity Factor and Class III Histone Deacetylase, Is an Essential Endogenous Apoptosis Inhibitor in Cardiac Myocytes
From the Cardiovascular Research Institute, Department of Cell Biology and Molecular Medicine (R.R.A., S.F.V., J.S.), University of Medicine and Dentistry of New Jersey, New Jersey Medical School, Newark; the Department of Physiology (L.A.K.), Faculty of Medicine, Institute of Cardiovascular Sciences, St. Boniface General Hospital Research Center, University of Manitoba, Winnipeg, Canada; and the Department of Molecular Biology and Pharmacology (S.I.), Washington University School of Medicine, St. Louis, Mo.
Correspondence to Junichi Sadoshima MD, PhD, Cardiovascular Research Institute, University of Medicine and Dentistry of New Jersey, New Jersey Medical School, 185 S Orange Ave, MSB G-609, Newark, NJ 07103. E-mail Sadoshju{at}umdnj.edu
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Abstract |
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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 Sir2
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 Sir2. 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 Sir2 inhibition causes apoptosis through p53. Overexpression of Sir2 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 Sir2 plays an essential role in mediating cell survival, whereas Sir2 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.
Key Words: histone deacetylase • Sir2 • p53 • apoptosis • cardiac hypertrophy
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Introduction |
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Increasing lines of evidence suggest that the balance between growth and death in cardiac myocytes plays an important role in determining long-term cardiac function in cardiovascular patients.1,2 Inhibition of cardiac myocyte apoptosis by broad spectrum caspase inhibitors protects the heart from development of congestive heart failure in experimental animals.3 Although stimulation of cardiac regeneration with adult stem cells may become an important arm of treatment for patients,4 either enhancing cell-protective mechanisms or preventing cardiac myocyte death would remain important goals in cardiovascular medicine.
Reversible acetylation of proteins regulates activity of signaling molecules mediating cell growth and death responses, including cardiac hypertrophy.5 Histone acetyltransferases (HATs) and histone deacetylases (HDACs) play an important role in regulating cardiac hypertrophy.5 Cardiac HDAC function drew the attention of many investigators because of the availability of HDAC inhibitors and their preceding application to cancer treatment.6 It has been shown that class I HDACs, including HDAC 1, 2, 3, and 8, positively regulate cardiac hypertrophy,7 whereas class II HDACs, including HDAC 4, 5, 7, and 9, negatively regulate cardiac hypertrophy.8 Although class I and II HDACs affect cell death in fibroblasts and neurons,6 regulation of cell death/survival of cardiac myocytes by HDACs is not well understood.
Yeast silent information regulator 2 (Sir2), an oxidized nicotinamide adenine dinucleotide (NAD+)–dependent HDAC and founding member of the HDAC class III family, functions in a wide array of cellular processes, including gene silencing, rDNA recombination, life span extension in response to caloric restriction, and DNA damage repair.9 Importantly, Sir2 deacetylates not only histones but also nonhistone substrates. For example, the mammalian homologue of Sir2 deacetylates p53 and the Forkhead box, class O family transcription factors, thereby regulating transcription and apoptotic cell death in mice and humans.10–13 Sir2 forms a complex with acetyltransferase p300/CBP associated factor and MyoD and retards skeletal muscle differentiation.14 Mice deficient in Sir2, the murine ortholog of Sir2, exhibit developmental abnormality in the heart and only infrequently survive postnatally,15 suggesting that Sir2 possesses important functions in the heart. Considering its function favoring longevity and cell survival and its unique coupling with the metabolic state of the cells, Sir2 is an attractive candidate for critically regulating cell survival in cardiac myocytes in response to stresses such as myocardial ischemia. However, the functional role of Sir2 in growth and death of cardiac myocytes remains unknown. The goal of this study was to test our hypothesis that the evolutionarily conserved longevity factor Sir2 exerts protective effects on cardiac myocytes.
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Materials and Methods |
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Materials
Anti-Sir2
antibody, anti-acetyl-p53 (Lys 320 and Lys 373/382) antibodies and anti-acetyl histone H3 and H4 antibodies were purchased from Upstate. Sirtinol and DEVD-CHO were from Calbiochem, and Trichostatin A (TSA) was from Sigma.
Plasmids
A plasmid harboring the gene for mouse Sir2 has been described previously.16 Dominant-negative Sir2 (DN-Sir2) was generated by mutating histidine 355 to alanine.10
Primary Culture of Neonatal Rat Ventricular Myocytes
Primary cultures of cardiac ventricular myocytes were prepared from 1-day-old Crl: (WI) BR-Wistar rats as described previously.17 Myocytes were cultured under serum-free conditions for 48 hours before experiments. Cell size and total protein content were obtained as described previously.17
Immunostaining
Cells were fixed in PBS containing 3.7% paraformaldehyde, permeabilized in PBS containing 0.3% Triton X, and blocked with 5% BSA. Immunostaining was performed using antimurine Sir2 antibody (1:100) and antisarcomeric myosin antibody (MF20; 1:500).
Reverse Transcription–Polymerase Chain Reaction
PCR primers for atrial natriuretic factor (ANF) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) have been described.17
Assays for Apoptosis
Histone-associated DNA fragments were quantitated by the Cell Death Detection ELISA (Roche).18
Immunoblot Analysis
Nuclear fractions were prepared as described.17 Whole-cell lysates were obtained using Lysis Buffer A, containing 150 mmol/L NaCl, 50 mmol/L Tris, pH 7.5, 0.1 mmol/L Na3VO4, 1 mmol/L NaF, 0.5 mmol 4-(2-aminoethyl)benzenesulfonyl fluoride hydrochloride, 1% Nonidet P-40, 0.1% sodium dodecyl sulfate, 0.5% deoxycholic acid, 0.5 µg/mL leupeptin, and 0.5 µg/mL aprotinin. For immunoblotting, the antimurine Sir2 antibody (1:10 000) was used.
Transfection and Luciferase Assays
One million cells were transfected with 4 µg of plasmid. The transcriptional activity of p53 was evaluated using p53-Luc (2 µg) containing 15 copies of specific p53 DNA-binding sequence (Stratagene). A plasmid carrying the ß-galactosidase gene (SV40–ß-gal; 1 µg) was cotransfected. We cotransfected 1 µg of expression plasmids consisting of pcDNA3.1, pcDNA3.1-Sir2, or pcDNA 3.1-DN-Sir2.
Adenoviral Vectors
Adenovirus harboring Sir2 (Ad-Sir2) or DN-Sir2 (Ad-DN-Sir2) was made using AdMax (Microbix). Adenovirus harboring X-linked inhibitor of apoptosis (Ad-XIAP) and that harboring temperature sensitive dominant-negative p53 (Ad-DN-p53) have been described previously.18,19
Dog Model of Left Ventricular Hypertrophy and Heart Failure
Dog models of left ventricular hypertrophy (LVH) and LVH plus heart failure were prepared as described previously.20 Dogs were obtained from Marshall BioResources (North Rose, NY). This study was approved by the Institutional Animal Care and Use Committee at New Jersey Medical School. Methods to generate these animal models are described in the online data supplement available at http://circres.ahajournals.org.
Statistical Analyses
Data are given as mean±SEM. Statistical analyses were performed using ANOVA and post hoc tests by the Tukey method. Significance was accepted at the P<0.05 level.
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Results |
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Sir2
Is Localized to the NucleusNuclear and cytoplasmic fractions prepared from neonatal rat cardiac myocytes were subjected to immunoblotting with antimurine Sir2
antibody. A 110-kDa protein was detected predominantly in the nuclear fraction (Figure 1A). Immunostaining indicated that Sir2 is localized to the nucleus in cardiac myocytes (Figure 1B).
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Sir2 Inhibition Induces Apoptosis, Whereas That of Class I and II HDACs Causes Hypertrophy in Cardiac Myocytes
To examine the function of Sir2, a class III HDAC, in cardiac myocytes, we used nicotinamide (NAM) and sirtinol, inhibitors of Sir2.21,22 We also treated myocytes with TSA, an inhibitor of class I and II HDACs.6 Treatment of cardiac myocytes with NAM or TSA increased acetylation of Histones 3 and 4,16 indicating that NAM and TSA inhibit HDACs (Figure 2A). Treatment with NAM for 48 hours caused dose-dependent increases in cell shrinkage and detachment, consistent with apoptosis (Figure 2B). Similar findings were also obtained with sirtinol. Cell death by NAM was not caused by increased osmolarity alone because the same concentrations of NaCl did not cause cell death. In contrast, TSA increased cell size, although it also caused death in some myocytes at high doses. Quantitative analyses indicated that NAM significantly reduced cell size and total protein content, whereas TSA exhibited opposite effects (Figure 2C and 2D). The increases in cell size and protein content by TSA were modest compared with those by FBS. RT-PCR analyses indicated that TSA and NAM significantly increased mRNA expression of ANF, a fetal-type gene upregulated by hypertrophic stimuli (Figure 2E). Interestingly, NAM more strongly upregulated ANF despite its negative effects on hypertrophy. Inhibition of class III HDACs may have a direct stimulatory effect on ANF transcription through acetylation of transcription factors.
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Inhibition of Endogenous Sir2 Induces Apoptosis in Cardiac Myocytes
We examined whether cardiac myocyte death by Sir2 inhibition is attributable to apoptosis. NAM and sirtinol significantly increased mononucleosomes and oligonucleosomes in the cytoplasm, a sensitive marker of apoptosis (Figure 3A). Although TSA also significantly increased nuclear fragmentation, it was not as strong as NAM (Figure 3A). Similar results were obtained using TUNEL staining (Figure 3B). NAM induced modest, but significant, cleavage of caspase-3, whereas TSA did not (Figure 3C). Sirtinol induced cleavage of caspase-3 almost equally as H2O2, an inducer of apoptosis. However, the effect of sirtinol was not as strong as that of chelerythrine, another inducer of apoptosis18 (Figure 3D). To examine whether activation of caspase-3 is involved in myocyte death by NAM and TSA, we examined the effect of XIAP and DEVD-CHO, inhibitors of caspases, on myocyte death. Ad-XIAP prevented increases in cytoplasmic accumulation of mononucleosomes and oligonucleosomes caused by NAM, whereas Ad-LacZ did not (Figure 3E). In contrast, Ad-XIAP did not affect cell death attributable to TSA (Figure 3F). Similar results were obtained using DEVD-CHO (Figure 3B). These results indicate that cardiac myocyte death induced by NAM is caspase dependent, whereas a modest increase in cell death by TSA is caspase independent.
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Overexpression of DN-Sir2 Enhances Cardiac Myocyte Apoptosis
To further confirm that inhibition of endogenous Sir2 stimulates cardiac myocyte apoptosis, we examined the effect of DN-Sir2. Transduction with Ad-DN-Sir2 induced a 7.7-fold increase in DN-Sir2 expression (data not shown). Ad-DN-Sir2 enhanced cell death of myocytes, determined by morphology and TUNEL staining, in serum-free cultures and in response to H2O2 (0.1 mmol/L) compared with Ad-LacZ (Figure 4A and 4B). The effect of DN-Sir2 was not as strong as NAM, possibly because of the relatively modest effectiveness of Sir2 (H355A) as a dominant negative. The level of cleaved caspase-3 was higher in DN-Sir2–transduced myocytes than in LacZ-transduced ones (Figure 4C). These results confirm that inhibition of HDAC activity of Sir2 enhances cardiac myocyte apoptosis.
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Inhibition of Sir2 Activates p53 Through Acetylation
We examined whether inhibition of endogenous Sir2 affects p53, a positive mediator of apoptosis, in cardiac myocytes. Treatment of myocytes with NAM dose-dependently increased p53-Luc activity (Figure 5A). The effect of NAM was specific to the p53 DNA-binding sequence because the control reporter gene did not respond to NAM. In contrast, TSA failed to activate p53-Luc (Figure 5A). To examine whether the status of acetylation in p53 is affected by NAM or TSA in cardiac myocytes, nuclear fractions were subjected to immunoblotting with antiacetylated p53 antibodies. Acetylation of p53 was increased at K373/382 (1.8-fold) and K320 (2.8-fold) in myocytes treated with NAM but not with TSA, suggesting that endogenous Sir2 plays an important role in inhibiting acetylation of p53 in cardiac myocytes (Figure 5B and 5C). Neither NAM nor TSA significantly changed the total amount of p53 (Figure 5D). Together, these results suggest that Sir2 inhibition increases acetylation of p53 and stimulates its transcriptional activity. Furthermore, Sir2 regulates acetylation of p53 more strongly than TSA-sensitive HDACs in cardiac myocytes.
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NAM-Induced Cardiac Myocyte Death Is Inhibited by DN-p53
We examined whether cardiac myocyte apoptosis resulting from Sir2 inhibition is mediated by p53. Cardiac myocytes were transduced with adenovirus-harboring temperature-sensitive DN-p53 and cultured at 39°C. DN-p53 slightly reduced cell shrinkage and nuclear fragmentation by serum starvation alone, but this did not reach statistical significance. In contrast, DN-p53 significantly reduced cell shrinkage and death caused by NAM (Figure 6A and 6B). Ad-LacZ failed to rescue NAM-induced cell shrinkage/death (Figure 6A and 6B). The temperature-sensitive DN-p53 construct works as a dominant negative only at high temperatures.19 Consistent with this notion, the cell death inhibitory effect of DN-p53 against NAM treatment was not observed at 32°C (Figure 6A). These results suggest that p53 plays an essential role in mediating myocyte apoptosis by NAM.
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Stimulation of Sir2 Blocks Cardiac Myocyte Apoptosis in Response to Serum Starvation
We examined whether overexpression of Sir2 prevents cardiac myocyte apoptosis induced by serum starvation. Transduction of myocytes with Ad-Sir2 (10 mois) caused a 4.7-fold increase in Sir2 protein expression. Overexpression of Sir2 in myocytes cultured under serum-free conditions increased cell size at days 2 and 3 and inhibited shrinkage and death of cardiac myocytes. Ad-LacZ transduction neither increased cell size nor prevented cell shrinkage (Figure 7A). The cell protective effects of Sir2 in serum-free culture became more prominent after longer incubation. After 7 days, many myocytes in a low-density culture exhibited shrinkage and death, whereas Sir2 transduction drastically reduced this effect (Figure 7B). Quantitative analyses showed that myocytes cultured in serum-free conditions for 48 hours had significantly increased cytoplasmic accumulation of mononucleosomes and oligonucleosomes compared with those in serum-containing medium. Overexpression of Sir2 inhibited increases in nuclear fragmentation induced by serum starvation, whereas transduction of Ad-LacZ did not (Figure 7C). Sir2 dose-dependently increased cell size 48 hours after transduction, whereas transduction of Ad-LacZ did not (Figure 7D). Sir2 significantly inhibited p53-Luc in myocytes cultured in serum-free conditions, whereas DN-Sir2 increased its activity (Figure 7E). It has been shown that pyruvate increases, whereas lactate decreases, the cellular NAD+/NADH ratio, thereby positively or negatively regulating the activity of endogenous Sir2.14 Consistent with this notion, pyruvate significantly inhibited cytoplasmic accumulation of mononucleosomes and oligonucleosomes induced by serum starvation, whereas lactate stimulated it (Figure 7F). These results suggest that the increased activity of either exogenous or endogenous Sir2 protects cardiac myocytes from apoptosis caused by serum starvation.
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Protein Expression of Sir2 Is Regulated in a Stimulus-Dependent Manner
We examined how protein expression of Sir2 is regulated in response to stresses. Sir2 expression was significantly downregulated in response to serum starvation. In contrast, other proapoptotic stimuli, including H2O2 and NAM, applied in the absence of serum, significantly upregulated Sir2 (Figure 8A).
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To examine whether protein expression of Sir2 in the heart is affected by pathologic stimuli in vivo, immunoblot analyses were conducted using left ventricular homogenates obtained from dogs with sham operation, compensated LVH, and LVH plus heart failure. Expression of a Sir2-like protein was significantly elevated in the failing hearts (Figure 8B and 8C).
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Discussion |
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Our results strongly suggest that endogenous Sir2
plays an essential role in mediating cell survival in cardiac myocytes. We speculate that decreases in Sir2 activity contribute to cardiac myocyte death in some pathologic conditions. For example, the HDAC activity of Sir2 critically depends on the nuclear content of NAD+. NAD+ is actively consumed when poly(ADP-ribose) polymerase (PARP) is activated by DNA strand breaks to participate in DNA repair.23 PARP activation also elevates NAM. Thus, DNA damage caused by oxidative stress, aging, myocardial ischemia/reperfusion, or genotoxic reagents should reduce the nuclear ratio of NAD+ to NAM through activation of PARP and negatively affect the activity of Sir2.24 Our results also showed that treatment with lactate, a procedure shown to decrease the ratio of NAD+ to NADH, thereby inhibiting Sir2, increased cardiac myocyte apoptosis.
Cardiac myocyte death caused by Sir2 inhibition is likely to be mediated at least in part by apoptosis because it was significantly, if not completely, suppressed by XIAP or DEVD-CHO. However, because activation of caspase-3 is relatively modest, the involvement of caspase-independent forms of cell death, such as necrosis, in myocyte death by Sir2 inhibition cannot be excluded at present. Interestingly, although inhibition of class I and II HDACs by TSA also induced a modest increase in nuclear fragmentation, the extent of cell death by TSA was smaller than that by NAM. Caspase-3 was not activated by TSA treatment, and XIAP and DEVD-CHO failed to inhibit TSA-induced nuclear fragmentation. Thus, modest cell death by TSA may not be apoptosis. Alternatively, TSA-induced cell death may be apoptosis but mediated by caspase-independent mechanisms. The transcriptional activity of p53 was stimulated by NAM but not by TSA, which is consistent with the notion that cell death induced by inhibition of class III, and that of class I and II HDACs, is mediated by distinct mechanisms.
It has been shown that the rare surviving Sir2-deficient mice have smaller bodies and organ size, as well as developmental abnormalities in several organs, including septal and valvular heart defects.15,25 Although some Sir2-deficient mice die in utero, whether or not apoptosis can be observed in cardiac myocytes has not been determined.15 Enhanced cell death in the heart may contribute to developmental abnormality in those mice.
Our results suggest that transcriptional activity of p53 is negatively regulated by Sir2 in cardiac myocytes. Acetylation as well as transcriptional activity of p53 were increased by NAM but not by TSA in cardiac myocytes. Although Sir2- and TSA-sensitive HDACs either deacetylate or promote degradation of p53 in fibroblasts,10,11,26 our results suggest that Sir2 plays a more important role in regulating p53 than class I and II HDACs in cardiac myocytes.
p53 regulation by Sir2 seems essential for cardiac myocyte survival because NAM-induced cardiac myocyte death and cytoplasmic accumulation of mononucleosomes and oligonucleosomes are strongly inhibited in the presence of DN-p53. p53 stimulates cardiac myocyte apoptosis via multiple mechanisms, including upregulation of the renin-angiotensin system,27 blockade of vacuolar proton ATPase,28 and activation of the mitochondrial death pathway.19 Development of congestive heart failure caused by telomere shortening is accompanied by p53 upregulation.29 However, it should be noted that p53 inhibition is not able to protect cardiac myocytes from apoptosis in response to some pathologic stimuli, possibly because of the presence of parallel proapoptotic mechanisms.30,31 Therefore, we speculate that the protective effect of Sir2 on cardiac myocytes should be manifested in response to a subset of pathologic stimuli, which promote cardiac myocyte apoptosis predominantly through p53. Sir2 deacetylates other transcription factors including MyoD14 and Forkhead transcription factors.12,13 It remains to be elucidated whether or not regulation of other transcription factors is also involved in the regulation of apoptosis/survival by Sir2 in cardiac myocytes.
Another important observation in this work is that increased dosage/activity of Sir2, either by adenovirus-mediated overexpression or by a procedure known to increase cellular NAD+/NADH ratio, decreased cardiac myocyte apoptosis caused by serum starvation. It has been shown recently that Sir2 activity is positively regulated by an intracellular redox sensitive mechanism, namely the ratio of NAD+ to NADH in skeletal muscle cells.14 Thus, a close relationship may exist between Sir2 and oxidative stress, which may contribute to cell survival regulation in response to serum starvation. We also found that expression of a Sir2 homologue is upregulated in failing dog hearts. It is expected that the oxidative environment of the failing heart would also enhance the activity of Sir2.14 It has been shown that resveratrol, a component of red wine, has cardioprotective effects, such as apoptosis inhibition.32 Interestingly, resveratrol has been identified recently as one of the strongest stimulators of Sir2.33 Thus, cardioprotective effects of resveratrol might be mediated by Sir2 activation. Together, our results suggest that Sir2 could mediate cell survival mechanisms in selected pathologic conditions, including starvation, aging, and heart failure, as well as in certain dietary conditions.
It has been shown recently that cardiac hypertrophy is dose-dependently inhibited by TSA.7 The authors speculated that transcription of antihypertrophic genes negatively regulated by class I HDACs are stimulated by TSA.7 However, our results suggest that TSA induces modest cardiac myocyte hypertrophy. Although the reason for the discrepancy is not clear at present, it has been shown that class II HDACs, such as HDAC 9, work as signal-responsive repressors of cardiac gene transcription mediating cardiac hypertrophy8 and that cAMP response element-binding protein and p300 stimulate hypertrophy through their HAT activity.34–36 Because TSA inhibits class I and class II HDACs,6 TSA-induced hypertrophy may be in part explained by action of TSA on class II HDACs. Alternatively, the hypertrophic action of TSA could have been obscured in the previous report because of preferential increases in cell death or concomitant reactivation of the antihypertrophic genes regulated by class I HDACs.7 Interestingly, our results suggest that overexpression of Sir2 causes increases in protein content as well as cardiac myocyte size. This result was surprising considering the known similarity of the effects of Sir2 to those caused by inhibiting insulin-like growth factor I signaling in Caenorhabditis elegans,37 which may negatively affect hypertrophy. At present, the signaling mechanism by which Sir2 induces hypertrophy is unknown. However, because it has been shown that Sir2 retards skeletal muscle differentiation, possibly by affecting the acetylation status of MyoD,14 it is possible that Sir2-induced hypertrophy is also in part mediated by regulation of transcription factors, which should be different from the one regulating ANF expression (Figure 2E). Together, our results suggest that cellular functions substantially differ among different subclasses of HDAC in cardiac myocytes. In this regard, application of HDAC inhibitors for treatment of cardiovascular disease should proceed with caution, taking their isoform-specific effects into important consideration.
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Acknowledgments |
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This work was supported by US Public Health Service grants HL 33107, HL 59139, HL67724, HL67727, HL69020, and HL 73048, and by American Heart Association grant 0340123N. L.A.K. is a Canada research chair in molecular cardiology. We thank Daniela Zablocki for critical reading of the manuscript and Dr Maha Abdellatif for helpful discussion.
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Footnotes |
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Original received June 9, 2004; revision received September 29, 2004; accepted September 30, 2004.
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