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Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation
Journal of Molecular and Cellular Cardiology 38 (2005) 163–174 www.elsevier.com/locate/yjmcc
Original Article
Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation Yasuhiro Maejima a, Susumu Adachi a,*, Kino Morikawa a, Hiroshi Ito b, Mitsuaki Isobe a a
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan b Second Department of Internal Medicine, Akita University, Japan Received 27 May 2004; received in revised form 17 September 2004; accepted 13 October 2004 Available online 08 December 2004
Abstract Two protein signaling systems, phosphorylation and S-nitrosylation, influence most aspects of cellular physiology. S-nitrosylation, which generates a nitrosothiol linkage on cysteine residues, is caused by nitric oxide (NO). NO is believed to act as an anti-apoptotic agent by inhibiting caspase activity in cardiomyocytes, but there is little direct evidence for this. We investigated whether apoptosis inhibition by NO involved S-nitrosylation of caspases in doxorubicin (DOX)-induced myocardial apoptosis. Cardiomyocytes were treated with 1 µmol/l of DOX to induce apoptosis. Pretreatment with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) reduced the apoptosis. This effect was attenuated by treatment with 100 µmol/l of mercury dichloride (HgCl2), which is an agent of denitrosylation. After 24 h DOX-treatment, SNAP reduced the increased caspase-3 activity by 63%, and this effect was reversed by treatment with HgCl2. Immunoblot analysis showed that accumulation of the cleaved caspase-3 protein, an active form that induces apoptosis was inhibited significantly by SNAP. To elucidate nitrosothiol formation on caspase-3 by NO, we did several experiments. First, we prepared an immunoprecipitate of caspase-3 and measured the concentration of NO released from the precipitated complex by HgCl2. Second, S-nitrosylated proteins, purified by immunoprecipitation of caspase-3, were biotinylated and the biotin concentration was estimated by immunoblotting. Third, dual immunofluorescent staining was done with antibodies for S-nitrosocysteine and caspase-3. Results showed that formation of nitrosothiol in caspase-3 in DOX-treated cardiomyocytes with SNAP was increased significantly compared with untreated cardiomyocytes. We reported here that exogenous NO produces an anti-apoptotic effect by suppression of caspase activity via S-nitrosylation in cardiomyocytes. © 2004 Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Apoptosis; S-nitrosylation; Cardiomyocyte; Caspase-3; Doxorubicin
1. Introduction Nitric oxide (NO) plays important regulatory roles in the neuronal, immune, and cardiovascular systems. NO regulates vascular tone, provides anti-inflammatory activity, and inhibits apoptosis, in part, by increasing the cellular concentration of cGMP. However, cGMP-independent cell signaling processes, including S-nitrosylation, are also affected by NO. Phosphorylation and S-nitrosylation, which are two important protein signaling systems, govern cell physiology. S-nitrosylation is a post-translational modification involving the attachment of NO to cysteine residues or transition metals [1]. Important S-nitrosylation targets are the caspase pro* Corresponding author. Tel.: +81-3-5803-5218; fax: +81-3-5803-0133. E-mail address: [email protected] (S. Adachi). 0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.10.012
teins. Some investigators have suggested that S-nitrosylation of the catalytic site cysteine in caspases serves as an on/off switch regulating caspase activity during apoptosis in endothelial cell, lymphocyte and trophoblast [2–4]. A family of caspases is a key regulator of apoptotic signaling pathway [5]. Generally, apoptotic caspases are categorized into initiator (caspase-8, -9) and executioner caspases (caspase-3, -6, -7) [6]. Inactive procaspases that exist as latent zymogens under normal conditions are cleaved into their active forms, composed of two large subunits and two small subunits, via other activated caspases in the apoptotic process. Active executioner caspase-3 can further cleave downstream substrates involved in apoptotic changes, such as poly(ADP-ribose) polymerase [7]. The cleavage of procaspases, an irreversible post-translational modification, has been used as an indicator of apoptosis. Considering that caspases are
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key mediators of the apoptotic process, identifying any regulatory modifications of these proteases is necessary to elucidate mechanisms of cellular balancing between survival and death. Heart failure is one of the major problems in cardiovascular disease. A major part of the pathological change in heart failure is caused by apoptosis, and modulating the factors that control apoptosis can prevent this component of cell death. Doxorubicin (DOX) is a broad-spectrum antitumor anthracycline antibiotic that is used widely for treatment of a variety of cancers, including severe leukemias, lymphomas, and solid tumors. The clinical use of DOX is limited because it strongly induces apoptotic cell death of cardiomyocytes [8]. However, it is unclear whether NO is involved in DOX-induced apoptosis in cardiomyocytes. Although investigation of the mechanism of anti-apoptosis in cardiomyocytes could lead to new treatments for heart failure, there are no reports that directly identify S-nitrosylation in endogenous procaspases in cardiomyocytes. In this study, we found that NO regulates the anti-apoptotic pathway by S-nitrosylation of caspase-3 in DOX-treated cardiomyocytes. We suggest that S-nitrosylation by NO is a novel cytoprotective reaction in cardiomyocytes.
2. Materials and methods 2.1. Cell culture and treatment Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were isolated, subjected to Percoll gradient centrifugation, and cultured in vitro as described previously [9]. The cardiomyocytes were incubated in Eagle’s minimum essential medium (Sigma Chemical Co., St. Louis, MO) supplemented with 5% calf serum (JRH Biosciences, Lenexa, KS) for 24 h at 37 °C. The cardiomyocytes were pretreated with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP, Dojindo, Kumamoto, Japan), for 90 min and for 15 min prior to treatment of 1 µmol/l DOX relevant dosage to induce myocardial apoptosis [8]. The cells were analyzed at various times. All experiments were done in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Animal Research Committee of the Tokyo Medical and Dental University. 2.2. Assay for apoptosis The percentage of cells undergoing apoptosis was calculated as the ratio of apoptotic cells to total cells. Apoptotic cardiomyocytes detach from the substratum; thus, the extent of apoptosis can be determined by counting floating and adherent cells with a cell counter (CDA-500, Sysmex, Long Grove, IL) [10,11]. For the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)
assay, floating cells were recovered from the medium by centrifugation and pooled with the adherent cells collected by trypsinization. Cells were spun down at 1000 × g onto polyL-lysine-coated slides, and TUNEL assays were done according to the manufacturer’s instructions with a commercially available kit for detecting end-labeled DNA (ApopTag, Oncor, Gaithersburg, MD) and with anti-digoxigenin-rhodamine (Roche Diagnostics, Indianapolis, IN). All experiments were repeated on at least three independent occasions with consistent results. 2.3. Flowcytometric analysis of propidium iodide (PI)-stained cells Apoptosis was assessed by quantifying hypodiploid nuclei undergoing DNA fragmentation via flowcytometric analysis as described previously [12]. This method is based on the observation that cells undergoing apoptosis when fixed in ethanol and stained with propidium iodide (PI) (Sigma Chemical Co.) have a hypodiploid quantity of DNA and localize in a broad area below the G0/G1 peak on a PI histogram. Ten thousand cells from each sample were counted with a FACSCalibur flowcytometer (Becton Dickinson, San Jose, CA). Gating was performed to exclude very small debris with 2 log units weaker PI staining than that observed in G0 cells. The percentage of cells in different cell cycle stages was assessed with Cell Quest software (Becton Dickinson). 2.4. Measurement of creatine phosphokinase concentration Although apoptotic cells maintain cell membrane integrity, necrosis usually involves cell lysis due to loss of integrity and spilling of the cell contents into the immediate environment, leading to leakage of the creatine phosphokinase (CPK) into the culture medium. Therefore, extracellular levels of CPK were determined as a means of detecting necrotic cells. The CPK concentration of the cardiomyocytes was measured according to the manufacturer’s instructions using the commercially available kit, CPK-test Wako kit (Wako Chemicals, Osaka, Japan). 2.5. Caspase-3 activity Caspase-3 assays were carried out using the CaspACE assay system (Promega, Madison, WI). The cells (5 × 106) were harvested and lyzed in 170 µl of the cell lysis buffer included with the kit, and protein concentrations were equalized for each condition. Subsequently, 100 µg of cell lysate was combined with an equal amount of substrate reaction buffer containing a caspase-3 colorimetric substrate, acetylDEVD-p-nitroaniline (Ac-DEVD-pNa). This mixture was incubated for 2 h at 37 °C, and then absorbance was measured with a plate reader (Ultramark, BIO-RAD, Hercules, CA).
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2.6. Antibodies and immunoblotting We used polyclonal antibodies for rabbit procaspase-3 (sc751, Santa Cruz Biotechnology, Santa Cruz, CA), cleaved caspase-3 (#9661S), c-jun NH2-terminal kinase (JNK, #9252) and phospho-JNK (epitope mapping at the amino acid residues around phospho-Thr 183 and phospho-Tyr 185 of human JNK, #9255S., Cell Signaling Technology, Beverly, MA). Electrophoresis was done on 10% SDS-polyacrylamide gels. Sample volumes were adjusted to give consistent gel staining with Coomassie brilliant blue. Gels were transferred to nitrocellulose membranes by semi-dry electrotransfer, and immunoblotting was done as described previously [13]. Densitometric analysis was done with NIH image software (National Institutes of Health, Bethesda, MD). 2.7. Cyclic GMP dependent protein kinase (cGK) activity assay cGK activity assays were carried out using the CycLex cGK Assay Kit (CycLex, Ina, Nagano, Japan). The cells (5 × 106) were harvested and lyzed in 50 µl of the cell lysis buffer and protein concentrations were equalized for each condition. Ten microliter of cell lysate was combined with 90 µl of kinase reaction buffer containing cGMP per well and incubated for 1 h at 30 °C. After washing wells, 100 µl of horseradish peroxydase-conjugated anti-phospho-G-kinase substrate antibody was added into each well and incubated for 1 h at room temperature. Subsequently, 100 µl of tetramethylbenzidine was added to each well and incubated for 15 min at room temperature, and then absorbance was measured at 450 nm with a plate reader. cGK activity was inhibited with NO-sensitive guanylate cyclase inhibitor, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (ODQ, 5 µmol/l, Calbiochem, La Jolla, CA) in the experiments. This concentration was already confirmed to inhibit cGK activity but not induce cytotoxicity [14,15]. 2.8. Measurement of peroxynitrite production Peroxynitrite production by cardiomyocytes was quantified using 2-[6-(4′-hydroxy) phenoxy-3H-xanthen-3-on-9yl]-benzoic acid (HPF, Daiichi Pure Chemicals, Tokyo, Japan) [16]. Cells were cultured in 96-well cell-culture plates (8 × 104 per well) with DOX and SNAP or medium control. The cells were incubated for 30 min at 37 °C with HPF (10 µmol/l). The fluorescent HPF activated by peroxynitrite was quantified in a fluorometer with an excitation wavelength of 490 nm and an emission wavelength of 515 nm. 2.9. Detection of S-nitrosylated caspase-3 by immunoblotting The procedure followed the protocol of Jaffrey et al. with modifications [17]. Briefly, freshly isolated neonatal rat car-
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diomyocytes were lyzed in 50 vol. of HENS buffer (25 mmol/l HEPES, pH 7.7, 0.1 mmol/l ethylenediamine tetraacetic acid, 0.01 mmol/l neocuproine, and 1% SDS) for 20 min and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was immunoprecipitated overnight with an anti-caspase-3 polyclonal antibody conjugated with preclearing protein A/G plus agarose beads. Immunoprecipitated caspase-3 zymogen was eluted in 100 mmol/l glycine (pH 3.0), to which 4 vol. of blocking buffer (9 vol. of HENS buffer plus 1 volume 25% SDS, adjusted to 20 mmol/l methylmethanthiosulfate (MMTS) with a 2 mol/l stock prepared in dimethylformamide) was added at 50 °C for 20 min with frequent vortexing. The MMTS was then removed by acetone precipitation (–20 °C) overnight. After resuspending the pellet in HENS buffer, 4 mmol/l N-[6-(biotinamido)hexyl]-3P(2P-pyridyldithio) propionamide and 1 mmol/l sodium ascorbate were added, and the mixture was incubated for 1 h at 25 °C in the dark. Gel electrophoresis was done under nonreducing conditions by SDS-PAGE. Biotinylated proteins were detected by using polyclonal antibody for rabbit biotin and horseradish peroxidase-linked streptavidin according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ). 2.10. Detection of S-nitrosylation of caspase-3 in vitro S-nitrosylated caspase-3 protein was measured with the Saville-Griess assay as described previously [18]. After immunoprecipitation, caspase-3 protein was released from protein A/G plus agarose beads by adding 100 mmol/l glycine (pH 3.0) on ice. The NO from the decomposition of S-nitrosylated caspase-3 protein was produced in the presence of 100 µmol/l mercury dichloride (HgCl2), which selectively removes NO from S-nitrosothiols for 10 min at 37 °C [4,19,20]. Samples were incubated for 5 min to allow the formation of the diazonium salt. Then, a 0.02% solution of N-(1-naphtyl) ethylenediamine dihydrochloride dissolved in 0.5 mol/l HCl was added, and azo dye formation was detected by spectrophotometry at 540 nm. 2.11. Immunostaining of S-nitrosylated proteins Immunofluorescent staining of S-nitrosylated proteins with polyclonal antibody for rabbit S-nitrosocysteine (487918, Calbiochem, San Diego, CA) and fluorescein-conjugated antirabbit IgG antibody (23827, Polysciences Inc., Warrington, PA) was done as described previously [10]. Distribution of caspase-3 protein in cardiomyocytes was identified using monoclonal antibody for mouse caspase-3 (BioVision, Mountain View, CA) with rhodamine-conjugated anti-mouse IgG antibody (23800, Polysciences Inc.). Immunofluorescent images were obtained with a laser scanning confocal microscope (LSM510, Carl Zeiss, Jena, Germany). As a negative control, fixed and permeabilized cells were preincubated with 0.8% HgCl2 for 1 h at 37 °C.
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Fig. 1. Evaluation of DOX-induced apoptosis in cardiomyocytes. A. Analysis by cell counting of cellular viability during exposure to 1 µmol/l DOX. B. Analysis of apoptosis induced by 1 µmol/l DOX. Percentages of apoptotic cells were calculated by the TUNEL method. C. Flowcytometric analysis to detect sub-G1 phase in cardiomyocytes. Values were calculated from three independent experiments. The mean ± S.D. is shown. *P < 0.01 compared to time 0.
2.12. Statistics Data are given as mean ± S.D. values. Differences were analyzed with one-way analysis of variance (ANOVA) and
post-hoc analysis was done with the Bonferroni/Dunn test. A P value of less than 0.01 was considered statistically significant.
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3. Results 3.1. NO prevents DOX-induced cardiomyocyte apoptosis To examine whether NO was the effector for DOXinduced apoptosis, cardiomyocytes were exposed to 1 mmol/l
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SNAP prior to DOX treatment. DOX alone reduced cell viability to 45.6 ± 16.0% after 24 h treatment. Pretreatment with 1 mmol/l SNAP increased cell viability to 70.9 ± 8.0% (Fig. 1A). Furthermore, DOX-induced apoptosis after 24 h, as evaluated by TUNEL assay, was significantly lower in cardiomyocytes pretreated with 1 mmol/l SNAP (Control, 9.2 ±
Fig. 2. Dose-dependent effect of SNAP for DOX-induced cardiomyocyte cell death. A. Measurement of peroxynitrite concentration in the culture medium of DOX-treated cardiomyocytes. B. Analysis by cell counting of cellular viability during exposure to DOX. C. Analysis of apoptosis induced by DOX. Percentages of apoptotic cells were calculated by the TUNEL method. D. Analysis of extracellular levels of CPK as a means of detecting necrotic cells. The mean ± S.D. is shown. *P < 0.01 compared to control.
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1.1%; SNAP, 9.2 ± 1.1%; DOX, 39.8 ± 1.9%; DOX + SNAP, 25.8 ± 3.6% (Fig. 1B)). For determination of cell cycle distribution, cells were stained with PI and analyzed by flowcytometry. Cells with hypodiploid DNA content (sub-G1) were considered the apoptotic fraction. The result of one representative experiment is shown in Fig. 1C. The proportion of cardiomyocytes treated with 1 mmol/l SNAP at the sub-G1 phase was significantly increased than in cardiomyocytes receiving no SNAP after 24 h of DOX-treatment (Control, 4.2 ± 1.9%; SNAP, 5.4 ± 0.8%; DOX, 13.4 ± 0.8%; DOX + SNAP, 9.2 ± 0.8%). These results demonstrate that DOX-induced apoptosis was time-dependent, and pretreatment with 1 mmol/l SNAP significantly protected cardiomyocytes from apoptosis.
3.2. High-dose SNAP induces peroxynitrite production and decreases the inhibitory effect for cell death in DOX-treated cardiomyocytes Dox is known to generate oxygen-derived free radicals [21]. And, NO and oxygen-derived free radicals produce peroxynitrite, which may induce cellular damages [22]. To ascertain whether peroxynitrite is formed by the combination of DOX and SNAP under these experimental conditions, we measured peroxynitrite concentration in the culture medium. Peroxynitrite production in DOX-treated cardiomyocytes significantly increased after stimulation with SNAP in a dosedependent manner (Fig. 2A). Next, to examine the dosedependent effect of SNAP for DOX-induced cardiomyocyte
Fig. 3. A. Caspase-3 activity (pmol/µl) in lysates from DOX-treated cardiomyocytes. Stimulated caspase-3 activity was lower in cell lysates from DOX + SNAP-treated cardiomyocytes (solid diamonds) than in cardiomyocytes treated with DOX only (solid squares). B. Changes in protein level of cleaved caspase3 in cardiomyocytes treated with DOX evaluated by immunoblot and densitometrical analysis. Values were calculated from three independent experiments. The mean ± S.D. is shown. *P < 0.01 compared to control. #P < 0.01 compared to DOX-only samples.
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cell death, cardiomyocytes were exposed to various concentrations of SNAP (0.1 and 10 mmol/l) prior to DOX-treatment. Contrary to the response of lower concentration of SNAP (0.1 and 1 mmol/l) for cardiomyocytes, pretreatment with a higher concentration of SNAP (5 and 10 mmol/l) decreased the inhibitory effect evaluated by viable cell counts and TUNEL positive cells (Fig. 2B, C). As shown in Fig. 2D, CPK levels, which is biochemical hallmark of necrosis were not increased in the condition of DOX-treated cardiomyocytes pretreated with SNAP at 0.1 and 1 mmol/l, as well as untreated cells. These results indicated that high-dose SNAP induces not only apoptosis but also necrosis to DOX-treated cardiomyocytes. The low concentration of SNAP (1 mmol/l), which was used in this experimental condition, revealed maximum inhibition of DOX-induced apoptosis. 3.3. NO inhibits caspase-3 activity in DOX-treated cardiomyocytes To ascertain whether the effect of NO as an anti-apoptotic agent resulted from blockade of caspase-3 activity, we measured caspase-3 catalytic activity in cardiomyocytes pretreated with SNAP and with DOX only. Treatment of cardiomyocytes with DOX caused a time-dependent caspase3 activity increase that was inhibited significantly by pretreatment with SNAP 37% of DOX-only activity after 24 h (Fig. 3A). To examine the effect of NO responsible for the caspase activity more directly, we did an immunoblot analysis for the cleavage of caspase-3 to its active 17 kDa subunit (Fig. 3B). The level of procaspase-3 accumulated in a time-dependent manner, but there was no difference between cardiomyocytes treated with DOX and cells treated with DOX and SNAP. We also confirmed that SNAP did not affect the mRNA level of procaspase-3 (data not shown). However, the active subunit for caspase-3 (cleaved caspase-3) was detected readily in DOX-treated cells but not in SNAP pretreated cells. These results suggested that NO inhibited caspase-3 catalytic activity in DOX-treated cardiomyocytes by suppressing the conversion of procaspase-3 to activated caspase-3. 3.4. The relationship between cGK activity and caspase-3 activity in DOX-treated cardiomyocytes NO is reported to activate cGK activity, which works as a cellular protection [23]. To exclude the involvement of cGK activation in this experimental condition, we evaluated caspase-3 activity with the treatment of ODQ. The cGK activity of DOX-treated cardiomyocytes increased in response to SNAP in a concentration dependent manner (Fig. 4A). Although an increase in caspase-3 activity by DOX was inhibited with SNAP treatment, cotreatment with ODQ and SNAP of DOX-treated cardiomyocytes did not revert caspase3 activity (Fig. 4B). These results indicate that cGK activity does not mediate the inhibitory effect of SNAP on caspase3 activity.
Fig. 4. The cGK activity and caspase-3 activity in DOX-treated cardiomyocytes. A. Analysis of cGK activity of DOX-treated cardiomyocytes pretreated with SNAP or DOX only. B. Caspase-3 activity (pmol/µl) in lysates from DOX-treated cardiomyocytes. Cotreatment with 5 µmol/l ODQ and SNAP of DOX-treated cardiomyocytes did not upregulate caspase-3 activity compared with treatment with SNAP alone for 24 h. The mean ± S.D. is shown. *P < 0.01 compared to control. #P < 0.01 compared to DOX-only samples.
3.5. Anti-apoptotic effect of NO is reversed by treatment with HgCl2 HgCl2 is known to selectively remove NO from S-nitrosothiols. Conversely, HgCl2 is an inhibitor of S-nitrosylation. The concentration of HgCl2 that we used did not induce cell death (data not shown). When cardiomyocytes were pretreated with both SNAP (1 mmol/l) and HgCl2 (100 µmol/l) before the addition of DOX, cell viability was reduced significantly after 36 h compared with cardiomyocytes treated with DOX and SNAP (Fig. 5A). Furthermore,
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DOX-induced apoptosis in cardiomyocytes treated with SNAP and HgCl2, as evaluated by TUNEL assay, was significantly higher than that of cells treated with SNAP only (Control, 9.5 ± 0.7%; DOX, 67.8 ± 5.5%; DOX + SNAP, 38.9 ± 4.5%; DOX + SNAP + HgCl2, 55.9 ± 2.5% (Fig. 5B)). The proportion of cardiomyocytes treated with SNAP and HgCl2 at the
sub-G1 phase was significantly higher than that of cells treated with SNAP only after 24 h of DOX-treatment (Control, 4.2 ± 1.9%; DOX, 13.4 ± 0.8%; DOX + SNAP, 9.2 ± 0.8%; DOX + SNAP + HgCl2, 12 ± 0.5% (Fig. 5C)). These results suggest that HgCl2 reduced the protective effect of SNAP in DOXtreated cardiomyocytes. Because SNAP reduced apoptosis and
Fig. 5. Anti-apoptotic effect of NO is reversed by HgCl2 (100 µmol/l) in DOX-treated cardiomyocytes. A. Analysis of cellular viability by cell-number counting of DOX-treated cells. B. Analysis of apoptosis by the TUNEL method of DOX-treated cells. C. Flowcytometric analysis to detect sub-G1 phase in cardiomyocytes. Values were calculated from three independent experiments. The mean ± S.D. is shown. #P < 0.01 compared to DOX-only samples. †P < 0.01 compared to cells treated with DOX + SNAP.
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S-nitrosylation. First, we treated cells with SNAP, lyzed the cells, and determined by Saville-Griess assay whether immunoprecipitated caspase-3 protein contained NO. Immunoprecipitated procaspase-3 from SNAP-treated cells released 2.5fold more NO (3.19 µmol/l) than did untreated cells (1.28 µmol/l)(Fig. 7A), confirming that the caspase-3 in cardiomyocytes is susceptible to nitrosylation in situ. Secondly, we measured S-nitrosylation of caspase-3 using the technique of Jaffrey et al. [17]. This analysis found that immunoprecipitated procaspase-3 from SNAP-treated cells was significantly nitrosylated compared with untreated cells. Cotreatment with ODQ, DOX, and SNAP have no effect on S-nitrosylation of immunoprecipitated procaspase-3, suggesting that S-nitrosylation of caspase-3 by a pathway, independent of cGMP (Fig. 7B). Finally, to confirm these results, we did an immunohistochemical examination. Dual staining with antibodies against caspase-3 and S-nitrosocysteine showed that S-nitrosylated molecules and caspase-3 colocalize in cardiomyocytes treated with DOX and SNAP (Fig. 7C). However, very little fluorescence signal was generated with an antibody against S-nitrosocysteine in cardiomyocytes treated with DOX only. Cotreatment with HgCl2 and SNAP in DOXtreated cardiomyocytes also generated little fluorescence signal. These results provide further support for the hypothesis that the inhibition of caspase-3 activity by S-nitrosylation is one of the major mechanisms by which NO inhibits apoptosis.
Fig. 6. A. Caspase-3 activity (pmol/µl) in lysates from DOX-treated cardiomyocytes. The inhibitory effect of SNAP on caspase-3 activity is reversed by incubation with HgCl2 (100 µmol/l) for 24 h (solid triangles). B. Changes in protein level of cleaved caspase-3 and phospho-JNK in cardiomyocytes treated with DOX evaluated by immunoblot analysis. The mean ± S.D. is shown. #P < 0.01 compared to DOX-only samples. †P < 0.01 compared to cells treated with DOX + SNAP.
inhibited the activation of caspase-3 in DOX-treated cardiomyocytes, we next examined whether S-nitrosylation was involved in the activation of caspase-3. Cotreatment with HgCl2 and SNAP of DOX-treated cardiomyocytes upregulated caspase-3 activity significantly by 1.9-fold compared with treatment with SNAP alone (Fig. 6A). Then, to examine whether NO also affects other apoptotic signal transductions, we examined the effect of NO for the phosphorylation of JNK. As shown in Fig. 6B, DOX activates phospho-JNK irrespective of HgCl2 cotreatment. On the other, cleaved caspase3 protein level was decreased by treatment with SNAP and reverted partially by cotreatment with HgCl2. These results indicate that HgCl2 cleaved S-nitrosothiols and thereby reduced the inhibitory effect of SNAP on caspase-3 activity. 3.6. Role of S-nitrosylation of caspase-3 in the anti-apoptotic effect of NO To clarify whether caspase-3 is nitrosylated by NO in cardiomyocytes, we did three experiments to examine
4. Discussion This study is the first to show that NO inhibits apoptosis in cardiomyocytes by S-nitrosylation of caspase-3. This conclusion was based on the findings that the NO donor, SNAP, was able to inhibit cardiomyocyte apoptosis induced by DOX and reduced caspase-3 activity; DOX-induced apoptosis was reversed by denitrosylation with addition of HgCl2; and immunoprecipitated caspase-3 in cardiomyocytes treated with the SNAP was heavily nitrosylated. The anti-apoptotic effects of NO in cardiomyocytes have been classified as cGMP-dependent and -independent. NO activates cGMP signaling through interaction with the heme group of guanylate cyclase. The production of cGMP causes activation of cGK that leads to and reveals the protective effect [24]. The anti-apoptotic effects of NO can also be mediated through a number of mechanisms independent of cGMP signaling such as activation of p38 MAPK [25], PI3-K/Akt [26], reduction of mitochondrial Ca2+ uptake [27], and a decrease in cyclin A-associated kinase activity [28]. We confirmed that cGK activity does not mediate the inhibitory effect of NO on caspase-3 activity and S-nitrosylation of caspase-3 by NO is a novel cGMP-independent anti-apoptotic mechanism in cardiomyocytes. S-nitrosylation is a ubiquitous post-translational protein modification with broad regulatory purview, analogous in that
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Fig. 7. A. NO content in immunoprecipitated caspase-3 from cardiomyocytes measured by Saville-Griess assay (*P < 0.01 vs. SNAP (–) group). B. S-nitrosylation of immunoprecipitated caspase-3 (*P < 0.01 vs. SNAP (–) group). C. Immunofluorescent staining for caspase-3 and S-nitrosylated protein observed with a laser scanning confocal microscope. Caspase-3 protein is highly expressed in DOX-treated cardiomyocytes (rhodamine, red), and co-expression of S-nitrosylated caspase-3 (FITC, green) is increased in cardiomyocytes treated with SNAP. Representative results are shown. All photographs were taken at the same magnification (100×).
respect to protein phosphorylation. However, the analysis of S-nitrosylated protein in situ has been impeded by substantial technical barriers, and there is little direct evidence for cellular protein S-nitrosylation. Recently, Jaffrey et al. [17] developed a new methodology that lowered the technical hurdles and significantly increased experimental access to NO biology. By using this technique, we confirmed directly that NO inhibits apoptosis by preventing the activation of caspase3 via S-nitrosylation. Pro-apoptotic caspase-3 consists of two subunits, p12 and p17, that form a heterodimer to build the active protease. Subunit p17 contains a reactive cysteine at position 163 in the
catalytic center that is essential for enzyme activity. NO inhibits the p17 subunit via S-nitrosylation of this critical cysteine [2]. Thus, the increased S-nitrosylation of the p17 subunit after exposure to NO donors contributes to the anti-apoptotic effect of NO in cardiomyocytes. Recent studies have found that growth factors such as insulin-like growth factor I, hepatocyte growth factor, and erythropoietin have a protective effect on cardiomyocytes in the failing heart [29–31]. Some investigators have suggested that these growth factors enhance endogenous NO production by various cells [32–34]. Exogenous NO production by non-myocytes surrounding cardiomyocytes, such as endothe-
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lial cells, macrophages, and smooth muscle cells, is an important mechanism regulating apoptosis in cardiomyocytes. Furthermore, some studies have found that various pharmacological interventions can restore NO synthesis and inhibit downstream caspase-3 activation in failing human myocardium [35,36]. Taken together, the protective effect of these growth factors may be due partially to enhancement of NO production in cells surrounding cardiomyocytes. The results of our study show these growth factors to be attractive as possible therapeutic agents for preventing apoptosis. Recent reports suggested that p21ras and thioredoxin (Trx) are also nitrosylated and work as anti-apoptotic molecules in endothelial cells [37,38]. An increased S-nitrosylation of p21ras enhances its activation and contributes to activation of MAP kinase signaling that may regulate apoptosis. Trx can exert its anti-apoptotic activity via redox regulatory activity by S-nitrosylation at cysteine 69. Schonhoff et al. [39] reported that nitrosylated cytochrome c released from mitochondria has a proapoptotic effect, but is unknown how this molecule works in cardiomyocytes. Therefore, it is worth to investigate the intra-cellular reaction of S-nitrosylation in cardiomyocyte, especially in apoptosis. It is known that the effects of NO on apoptosis are not only inhibitory as shown in this study but also stimulatory [40]. It has been the subject of discussion as to why the same molecule should perform both these opposite tasks and it is thought that the biological conditions, such as the redox state, concentration, exposure time and the combination with oxygen, superoxide and other molecules, determine the net effects of NO on apoptosis [41]. In this experimental condition, these factors to the cells may affect S-nitrosylation of caspase-3. Also, NO is implicated in both apoptotic and necrotic cell death depending on the NO chemistry and the cellular biological redox state [41]. DOX is known to generate oxygenderived free radicals, and NO and oxygen-derived free radicals produce peroxynitrite, which may induce cellular damage. In the present study, we showed that high-dose SNAP induces apoptosis and also necrosis in DOX-treated cardiomyocytes, and peroxynitrite production significantly increased after stimulation with SNAP in dose-dependent manner. Taken together, a large quantity of peroxynitrite formed by the combination of DOX and high-dose NO may play a detrimental role for cardiomyocytes. In conclusion, exogenous NO induces S-nitrosylation of caspase-3, blocks its activation, and thereby partially inhibits DOX-induced apoptosis in cardiomyocytes. The ability of NO to inhibit downstream caspase-3 means that NO may be able to rescue cardiomyocytes from apoptosis even after the caspase cascade has been activated. Depletion of NO from non-myocytes or cardiomyocytes increases caspase-3 activity and might be associated with progressive ventricular remodeling and heart failure. The bioavailability of NO production might inhibit caspase-3 activation and prevent cardiomyocyte loss and remodeling in heart failure. Although, cardiomyocytes occupy over 75% of the structural space of the
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heart, the remaining non-myocytes consist cardiac fibroblasts, endothelial cells, coronary smooth muscle cells, and so on. To confirm whether supplementation of NO is beneficial for DOX-induced heart failure, further study must await, such as the experiments using non-myocytes and in vivo study.
Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan; a Research Grant for Diseases from the Ministry of Health and Welfare of Japan; and a Grant from the Japan Cardiovascular Research Foundation.
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Original Article
Nitric oxide inhibits myocardial apoptosis by preventing caspase-3 activity via S-nitrosylation Yasuhiro Maejima a, Susumu Adachi a,*, Kino Morikawa a, Hiroshi Ito b, Mitsuaki Isobe a a
Department of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan b Second Department of Internal Medicine, Akita University, Japan Received 27 May 2004; received in revised form 17 September 2004; accepted 13 October 2004 Available online 08 December 2004
Abstract Two protein signaling systems, phosphorylation and S-nitrosylation, influence most aspects of cellular physiology. S-nitrosylation, which generates a nitrosothiol linkage on cysteine residues, is caused by nitric oxide (NO). NO is believed to act as an anti-apoptotic agent by inhibiting caspase activity in cardiomyocytes, but there is little direct evidence for this. We investigated whether apoptosis inhibition by NO involved S-nitrosylation of caspases in doxorubicin (DOX)-induced myocardial apoptosis. Cardiomyocytes were treated with 1 µmol/l of DOX to induce apoptosis. Pretreatment with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP) reduced the apoptosis. This effect was attenuated by treatment with 100 µmol/l of mercury dichloride (HgCl2), which is an agent of denitrosylation. After 24 h DOX-treatment, SNAP reduced the increased caspase-3 activity by 63%, and this effect was reversed by treatment with HgCl2. Immunoblot analysis showed that accumulation of the cleaved caspase-3 protein, an active form that induces apoptosis was inhibited significantly by SNAP. To elucidate nitrosothiol formation on caspase-3 by NO, we did several experiments. First, we prepared an immunoprecipitate of caspase-3 and measured the concentration of NO released from the precipitated complex by HgCl2. Second, S-nitrosylated proteins, purified by immunoprecipitation of caspase-3, were biotinylated and the biotin concentration was estimated by immunoblotting. Third, dual immunofluorescent staining was done with antibodies for S-nitrosocysteine and caspase-3. Results showed that formation of nitrosothiol in caspase-3 in DOX-treated cardiomyocytes with SNAP was increased significantly compared with untreated cardiomyocytes. We reported here that exogenous NO produces an anti-apoptotic effect by suppression of caspase activity via S-nitrosylation in cardiomyocytes. © 2004 Elsevier Ltd. All rights reserved. Keywords: Nitric oxide; Apoptosis; S-nitrosylation; Cardiomyocyte; Caspase-3; Doxorubicin
1. Introduction Nitric oxide (NO) plays important regulatory roles in the neuronal, immune, and cardiovascular systems. NO regulates vascular tone, provides anti-inflammatory activity, and inhibits apoptosis, in part, by increasing the cellular concentration of cGMP. However, cGMP-independent cell signaling processes, including S-nitrosylation, are also affected by NO. Phosphorylation and S-nitrosylation, which are two important protein signaling systems, govern cell physiology. S-nitrosylation is a post-translational modification involving the attachment of NO to cysteine residues or transition metals [1]. Important S-nitrosylation targets are the caspase pro* Corresponding author. Tel.: +81-3-5803-5218; fax: +81-3-5803-0133. E-mail address: [email protected] (S. Adachi). 0022-2828/$ - see front matter © 2004 Elsevier Ltd. All rights reserved. doi:10.1016/j.yjmcc.2004.10.012
teins. Some investigators have suggested that S-nitrosylation of the catalytic site cysteine in caspases serves as an on/off switch regulating caspase activity during apoptosis in endothelial cell, lymphocyte and trophoblast [2–4]. A family of caspases is a key regulator of apoptotic signaling pathway [5]. Generally, apoptotic caspases are categorized into initiator (caspase-8, -9) and executioner caspases (caspase-3, -6, -7) [6]. Inactive procaspases that exist as latent zymogens under normal conditions are cleaved into their active forms, composed of two large subunits and two small subunits, via other activated caspases in the apoptotic process. Active executioner caspase-3 can further cleave downstream substrates involved in apoptotic changes, such as poly(ADP-ribose) polymerase [7]. The cleavage of procaspases, an irreversible post-translational modification, has been used as an indicator of apoptosis. Considering that caspases are
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key mediators of the apoptotic process, identifying any regulatory modifications of these proteases is necessary to elucidate mechanisms of cellular balancing between survival and death. Heart failure is one of the major problems in cardiovascular disease. A major part of the pathological change in heart failure is caused by apoptosis, and modulating the factors that control apoptosis can prevent this component of cell death. Doxorubicin (DOX) is a broad-spectrum antitumor anthracycline antibiotic that is used widely for treatment of a variety of cancers, including severe leukemias, lymphomas, and solid tumors. The clinical use of DOX is limited because it strongly induces apoptotic cell death of cardiomyocytes [8]. However, it is unclear whether NO is involved in DOX-induced apoptosis in cardiomyocytes. Although investigation of the mechanism of anti-apoptosis in cardiomyocytes could lead to new treatments for heart failure, there are no reports that directly identify S-nitrosylation in endogenous procaspases in cardiomyocytes. In this study, we found that NO regulates the anti-apoptotic pathway by S-nitrosylation of caspase-3 in DOX-treated cardiomyocytes. We suggest that S-nitrosylation by NO is a novel cytoprotective reaction in cardiomyocytes.
2. Materials and methods 2.1. Cell culture and treatment Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were isolated, subjected to Percoll gradient centrifugation, and cultured in vitro as described previously [9]. The cardiomyocytes were incubated in Eagle’s minimum essential medium (Sigma Chemical Co., St. Louis, MO) supplemented with 5% calf serum (JRH Biosciences, Lenexa, KS) for 24 h at 37 °C. The cardiomyocytes were pretreated with an NO donor, S-nitroso-N-acetyl-penicillamine (SNAP, Dojindo, Kumamoto, Japan), for 90 min and for 15 min prior to treatment of 1 µmol/l DOX relevant dosage to induce myocardial apoptosis [8]. The cells were analyzed at various times. All experiments were done in accordance with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996) and were approved by the Animal Research Committee of the Tokyo Medical and Dental University. 2.2. Assay for apoptosis The percentage of cells undergoing apoptosis was calculated as the ratio of apoptotic cells to total cells. Apoptotic cardiomyocytes detach from the substratum; thus, the extent of apoptosis can be determined by counting floating and adherent cells with a cell counter (CDA-500, Sysmex, Long Grove, IL) [10,11]. For the in situ terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL)
assay, floating cells were recovered from the medium by centrifugation and pooled with the adherent cells collected by trypsinization. Cells were spun down at 1000 × g onto polyL-lysine-coated slides, and TUNEL assays were done according to the manufacturer’s instructions with a commercially available kit for detecting end-labeled DNA (ApopTag, Oncor, Gaithersburg, MD) and with anti-digoxigenin-rhodamine (Roche Diagnostics, Indianapolis, IN). All experiments were repeated on at least three independent occasions with consistent results. 2.3. Flowcytometric analysis of propidium iodide (PI)-stained cells Apoptosis was assessed by quantifying hypodiploid nuclei undergoing DNA fragmentation via flowcytometric analysis as described previously [12]. This method is based on the observation that cells undergoing apoptosis when fixed in ethanol and stained with propidium iodide (PI) (Sigma Chemical Co.) have a hypodiploid quantity of DNA and localize in a broad area below the G0/G1 peak on a PI histogram. Ten thousand cells from each sample were counted with a FACSCalibur flowcytometer (Becton Dickinson, San Jose, CA). Gating was performed to exclude very small debris with 2 log units weaker PI staining than that observed in G0 cells. The percentage of cells in different cell cycle stages was assessed with Cell Quest software (Becton Dickinson). 2.4. Measurement of creatine phosphokinase concentration Although apoptotic cells maintain cell membrane integrity, necrosis usually involves cell lysis due to loss of integrity and spilling of the cell contents into the immediate environment, leading to leakage of the creatine phosphokinase (CPK) into the culture medium. Therefore, extracellular levels of CPK were determined as a means of detecting necrotic cells. The CPK concentration of the cardiomyocytes was measured according to the manufacturer’s instructions using the commercially available kit, CPK-test Wako kit (Wako Chemicals, Osaka, Japan). 2.5. Caspase-3 activity Caspase-3 assays were carried out using the CaspACE assay system (Promega, Madison, WI). The cells (5 × 106) were harvested and lyzed in 170 µl of the cell lysis buffer included with the kit, and protein concentrations were equalized for each condition. Subsequently, 100 µg of cell lysate was combined with an equal amount of substrate reaction buffer containing a caspase-3 colorimetric substrate, acetylDEVD-p-nitroaniline (Ac-DEVD-pNa). This mixture was incubated for 2 h at 37 °C, and then absorbance was measured with a plate reader (Ultramark, BIO-RAD, Hercules, CA).
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2.6. Antibodies and immunoblotting We used polyclonal antibodies for rabbit procaspase-3 (sc751, Santa Cruz Biotechnology, Santa Cruz, CA), cleaved caspase-3 (#9661S), c-jun NH2-terminal kinase (JNK, #9252) and phospho-JNK (epitope mapping at the amino acid residues around phospho-Thr 183 and phospho-Tyr 185 of human JNK, #9255S., Cell Signaling Technology, Beverly, MA). Electrophoresis was done on 10% SDS-polyacrylamide gels. Sample volumes were adjusted to give consistent gel staining with Coomassie brilliant blue. Gels were transferred to nitrocellulose membranes by semi-dry electrotransfer, and immunoblotting was done as described previously [13]. Densitometric analysis was done with NIH image software (National Institutes of Health, Bethesda, MD). 2.7. Cyclic GMP dependent protein kinase (cGK) activity assay cGK activity assays were carried out using the CycLex cGK Assay Kit (CycLex, Ina, Nagano, Japan). The cells (5 × 106) were harvested and lyzed in 50 µl of the cell lysis buffer and protein concentrations were equalized for each condition. Ten microliter of cell lysate was combined with 90 µl of kinase reaction buffer containing cGMP per well and incubated for 1 h at 30 °C. After washing wells, 100 µl of horseradish peroxydase-conjugated anti-phospho-G-kinase substrate antibody was added into each well and incubated for 1 h at room temperature. Subsequently, 100 µl of tetramethylbenzidine was added to each well and incubated for 15 min at room temperature, and then absorbance was measured at 450 nm with a plate reader. cGK activity was inhibited with NO-sensitive guanylate cyclase inhibitor, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxaline-1-one (ODQ, 5 µmol/l, Calbiochem, La Jolla, CA) in the experiments. This concentration was already confirmed to inhibit cGK activity but not induce cytotoxicity [14,15]. 2.8. Measurement of peroxynitrite production Peroxynitrite production by cardiomyocytes was quantified using 2-[6-(4′-hydroxy) phenoxy-3H-xanthen-3-on-9yl]-benzoic acid (HPF, Daiichi Pure Chemicals, Tokyo, Japan) [16]. Cells were cultured in 96-well cell-culture plates (8 × 104 per well) with DOX and SNAP or medium control. The cells were incubated for 30 min at 37 °C with HPF (10 µmol/l). The fluorescent HPF activated by peroxynitrite was quantified in a fluorometer with an excitation wavelength of 490 nm and an emission wavelength of 515 nm. 2.9. Detection of S-nitrosylated caspase-3 by immunoblotting The procedure followed the protocol of Jaffrey et al. with modifications [17]. Briefly, freshly isolated neonatal rat car-
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diomyocytes were lyzed in 50 vol. of HENS buffer (25 mmol/l HEPES, pH 7.7, 0.1 mmol/l ethylenediamine tetraacetic acid, 0.01 mmol/l neocuproine, and 1% SDS) for 20 min and then centrifuged at 12,000 × g for 15 min at 4 °C. The supernatant was immunoprecipitated overnight with an anti-caspase-3 polyclonal antibody conjugated with preclearing protein A/G plus agarose beads. Immunoprecipitated caspase-3 zymogen was eluted in 100 mmol/l glycine (pH 3.0), to which 4 vol. of blocking buffer (9 vol. of HENS buffer plus 1 volume 25% SDS, adjusted to 20 mmol/l methylmethanthiosulfate (MMTS) with a 2 mol/l stock prepared in dimethylformamide) was added at 50 °C for 20 min with frequent vortexing. The MMTS was then removed by acetone precipitation (–20 °C) overnight. After resuspending the pellet in HENS buffer, 4 mmol/l N-[6-(biotinamido)hexyl]-3P(2P-pyridyldithio) propionamide and 1 mmol/l sodium ascorbate were added, and the mixture was incubated for 1 h at 25 °C in the dark. Gel electrophoresis was done under nonreducing conditions by SDS-PAGE. Biotinylated proteins were detected by using polyclonal antibody for rabbit biotin and horseradish peroxidase-linked streptavidin according to the manufacturer’s protocol (Amersham Biosciences, Piscataway, NJ). 2.10. Detection of S-nitrosylation of caspase-3 in vitro S-nitrosylated caspase-3 protein was measured with the Saville-Griess assay as described previously [18]. After immunoprecipitation, caspase-3 protein was released from protein A/G plus agarose beads by adding 100 mmol/l glycine (pH 3.0) on ice. The NO from the decomposition of S-nitrosylated caspase-3 protein was produced in the presence of 100 µmol/l mercury dichloride (HgCl2), which selectively removes NO from S-nitrosothiols for 10 min at 37 °C [4,19,20]. Samples were incubated for 5 min to allow the formation of the diazonium salt. Then, a 0.02% solution of N-(1-naphtyl) ethylenediamine dihydrochloride dissolved in 0.5 mol/l HCl was added, and azo dye formation was detected by spectrophotometry at 540 nm. 2.11. Immunostaining of S-nitrosylated proteins Immunofluorescent staining of S-nitrosylated proteins with polyclonal antibody for rabbit S-nitrosocysteine (487918, Calbiochem, San Diego, CA) and fluorescein-conjugated antirabbit IgG antibody (23827, Polysciences Inc., Warrington, PA) was done as described previously [10]. Distribution of caspase-3 protein in cardiomyocytes was identified using monoclonal antibody for mouse caspase-3 (BioVision, Mountain View, CA) with rhodamine-conjugated anti-mouse IgG antibody (23800, Polysciences Inc.). Immunofluorescent images were obtained with a laser scanning confocal microscope (LSM510, Carl Zeiss, Jena, Germany). As a negative control, fixed and permeabilized cells were preincubated with 0.8% HgCl2 for 1 h at 37 °C.
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Fig. 1. Evaluation of DOX-induced apoptosis in cardiomyocytes. A. Analysis by cell counting of cellular viability during exposure to 1 µmol/l DOX. B. Analysis of apoptosis induced by 1 µmol/l DOX. Percentages of apoptotic cells were calculated by the TUNEL method. C. Flowcytometric analysis to detect sub-G1 phase in cardiomyocytes. Values were calculated from three independent experiments. The mean ± S.D. is shown. *P < 0.01 compared to time 0.
2.12. Statistics Data are given as mean ± S.D. values. Differences were analyzed with one-way analysis of variance (ANOVA) and
post-hoc analysis was done with the Bonferroni/Dunn test. A P value of less than 0.01 was considered statistically significant.
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3. Results 3.1. NO prevents DOX-induced cardiomyocyte apoptosis To examine whether NO was the effector for DOXinduced apoptosis, cardiomyocytes were exposed to 1 mmol/l
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SNAP prior to DOX treatment. DOX alone reduced cell viability to 45.6 ± 16.0% after 24 h treatment. Pretreatment with 1 mmol/l SNAP increased cell viability to 70.9 ± 8.0% (Fig. 1A). Furthermore, DOX-induced apoptosis after 24 h, as evaluated by TUNEL assay, was significantly lower in cardiomyocytes pretreated with 1 mmol/l SNAP (Control, 9.2 ±
Fig. 2. Dose-dependent effect of SNAP for DOX-induced cardiomyocyte cell death. A. Measurement of peroxynitrite concentration in the culture medium of DOX-treated cardiomyocytes. B. Analysis by cell counting of cellular viability during exposure to DOX. C. Analysis of apoptosis induced by DOX. Percentages of apoptotic cells were calculated by the TUNEL method. D. Analysis of extracellular levels of CPK as a means of detecting necrotic cells. The mean ± S.D. is shown. *P < 0.01 compared to control.
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1.1%; SNAP, 9.2 ± 1.1%; DOX, 39.8 ± 1.9%; DOX + SNAP, 25.8 ± 3.6% (Fig. 1B)). For determination of cell cycle distribution, cells were stained with PI and analyzed by flowcytometry. Cells with hypodiploid DNA content (sub-G1) were considered the apoptotic fraction. The result of one representative experiment is shown in Fig. 1C. The proportion of cardiomyocytes treated with 1 mmol/l SNAP at the sub-G1 phase was significantly increased than in cardiomyocytes receiving no SNAP after 24 h of DOX-treatment (Control, 4.2 ± 1.9%; SNAP, 5.4 ± 0.8%; DOX, 13.4 ± 0.8%; DOX + SNAP, 9.2 ± 0.8%). These results demonstrate that DOX-induced apoptosis was time-dependent, and pretreatment with 1 mmol/l SNAP significantly protected cardiomyocytes from apoptosis.
3.2. High-dose SNAP induces peroxynitrite production and decreases the inhibitory effect for cell death in DOX-treated cardiomyocytes Dox is known to generate oxygen-derived free radicals [21]. And, NO and oxygen-derived free radicals produce peroxynitrite, which may induce cellular damages [22]. To ascertain whether peroxynitrite is formed by the combination of DOX and SNAP under these experimental conditions, we measured peroxynitrite concentration in the culture medium. Peroxynitrite production in DOX-treated cardiomyocytes significantly increased after stimulation with SNAP in a dosedependent manner (Fig. 2A). Next, to examine the dosedependent effect of SNAP for DOX-induced cardiomyocyte
Fig. 3. A. Caspase-3 activity (pmol/µl) in lysates from DOX-treated cardiomyocytes. Stimulated caspase-3 activity was lower in cell lysates from DOX + SNAP-treated cardiomyocytes (solid diamonds) than in cardiomyocytes treated with DOX only (solid squares). B. Changes in protein level of cleaved caspase3 in cardiomyocytes treated with DOX evaluated by immunoblot and densitometrical analysis. Values were calculated from three independent experiments. The mean ± S.D. is shown. *P < 0.01 compared to control. #P < 0.01 compared to DOX-only samples.
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cell death, cardiomyocytes were exposed to various concentrations of SNAP (0.1 and 10 mmol/l) prior to DOX-treatment. Contrary to the response of lower concentration of SNAP (0.1 and 1 mmol/l) for cardiomyocytes, pretreatment with a higher concentration of SNAP (5 and 10 mmol/l) decreased the inhibitory effect evaluated by viable cell counts and TUNEL positive cells (Fig. 2B, C). As shown in Fig. 2D, CPK levels, which is biochemical hallmark of necrosis were not increased in the condition of DOX-treated cardiomyocytes pretreated with SNAP at 0.1 and 1 mmol/l, as well as untreated cells. These results indicated that high-dose SNAP induces not only apoptosis but also necrosis to DOX-treated cardiomyocytes. The low concentration of SNAP (1 mmol/l), which was used in this experimental condition, revealed maximum inhibition of DOX-induced apoptosis. 3.3. NO inhibits caspase-3 activity in DOX-treated cardiomyocytes To ascertain whether the effect of NO as an anti-apoptotic agent resulted from blockade of caspase-3 activity, we measured caspase-3 catalytic activity in cardiomyocytes pretreated with SNAP and with DOX only. Treatment of cardiomyocytes with DOX caused a time-dependent caspase3 activity increase that was inhibited significantly by pretreatment with SNAP 37% of DOX-only activity after 24 h (Fig. 3A). To examine the effect of NO responsible for the caspase activity more directly, we did an immunoblot analysis for the cleavage of caspase-3 to its active 17 kDa subunit (Fig. 3B). The level of procaspase-3 accumulated in a time-dependent manner, but there was no difference between cardiomyocytes treated with DOX and cells treated with DOX and SNAP. We also confirmed that SNAP did not affect the mRNA level of procaspase-3 (data not shown). However, the active subunit for caspase-3 (cleaved caspase-3) was detected readily in DOX-treated cells but not in SNAP pretreated cells. These results suggested that NO inhibited caspase-3 catalytic activity in DOX-treated cardiomyocytes by suppressing the conversion of procaspase-3 to activated caspase-3. 3.4. The relationship between cGK activity and caspase-3 activity in DOX-treated cardiomyocytes NO is reported to activate cGK activity, which works as a cellular protection [23]. To exclude the involvement of cGK activation in this experimental condition, we evaluated caspase-3 activity with the treatment of ODQ. The cGK activity of DOX-treated cardiomyocytes increased in response to SNAP in a concentration dependent manner (Fig. 4A). Although an increase in caspase-3 activity by DOX was inhibited with SNAP treatment, cotreatment with ODQ and SNAP of DOX-treated cardiomyocytes did not revert caspase3 activity (Fig. 4B). These results indicate that cGK activity does not mediate the inhibitory effect of SNAP on caspase3 activity.
Fig. 4. The cGK activity and caspase-3 activity in DOX-treated cardiomyocytes. A. Analysis of cGK activity of DOX-treated cardiomyocytes pretreated with SNAP or DOX only. B. Caspase-3 activity (pmol/µl) in lysates from DOX-treated cardiomyocytes. Cotreatment with 5 µmol/l ODQ and SNAP of DOX-treated cardiomyocytes did not upregulate caspase-3 activity compared with treatment with SNAP alone for 24 h. The mean ± S.D. is shown. *P < 0.01 compared to control. #P < 0.01 compared to DOX-only samples.
3.5. Anti-apoptotic effect of NO is reversed by treatment with HgCl2 HgCl2 is known to selectively remove NO from S-nitrosothiols. Conversely, HgCl2 is an inhibitor of S-nitrosylation. The concentration of HgCl2 that we used did not induce cell death (data not shown). When cardiomyocytes were pretreated with both SNAP (1 mmol/l) and HgCl2 (100 µmol/l) before the addition of DOX, cell viability was reduced significantly after 36 h compared with cardiomyocytes treated with DOX and SNAP (Fig. 5A). Furthermore,
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DOX-induced apoptosis in cardiomyocytes treated with SNAP and HgCl2, as evaluated by TUNEL assay, was significantly higher than that of cells treated with SNAP only (Control, 9.5 ± 0.7%; DOX, 67.8 ± 5.5%; DOX + SNAP, 38.9 ± 4.5%; DOX + SNAP + HgCl2, 55.9 ± 2.5% (Fig. 5B)). The proportion of cardiomyocytes treated with SNAP and HgCl2 at the
sub-G1 phase was significantly higher than that of cells treated with SNAP only after 24 h of DOX-treatment (Control, 4.2 ± 1.9%; DOX, 13.4 ± 0.8%; DOX + SNAP, 9.2 ± 0.8%; DOX + SNAP + HgCl2, 12 ± 0.5% (Fig. 5C)). These results suggest that HgCl2 reduced the protective effect of SNAP in DOXtreated cardiomyocytes. Because SNAP reduced apoptosis and
Fig. 5. Anti-apoptotic effect of NO is reversed by HgCl2 (100 µmol/l) in DOX-treated cardiomyocytes. A. Analysis of cellular viability by cell-number counting of DOX-treated cells. B. Analysis of apoptosis by the TUNEL method of DOX-treated cells. C. Flowcytometric analysis to detect sub-G1 phase in cardiomyocytes. Values were calculated from three independent experiments. The mean ± S.D. is shown. #P < 0.01 compared to DOX-only samples. †P < 0.01 compared to cells treated with DOX + SNAP.
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S-nitrosylation. First, we treated cells with SNAP, lyzed the cells, and determined by Saville-Griess assay whether immunoprecipitated caspase-3 protein contained NO. Immunoprecipitated procaspase-3 from SNAP-treated cells released 2.5fold more NO (3.19 µmol/l) than did untreated cells (1.28 µmol/l)(Fig. 7A), confirming that the caspase-3 in cardiomyocytes is susceptible to nitrosylation in situ. Secondly, we measured S-nitrosylation of caspase-3 using the technique of Jaffrey et al. [17]. This analysis found that immunoprecipitated procaspase-3 from SNAP-treated cells was significantly nitrosylated compared with untreated cells. Cotreatment with ODQ, DOX, and SNAP have no effect on S-nitrosylation of immunoprecipitated procaspase-3, suggesting that S-nitrosylation of caspase-3 by a pathway, independent of cGMP (Fig. 7B). Finally, to confirm these results, we did an immunohistochemical examination. Dual staining with antibodies against caspase-3 and S-nitrosocysteine showed that S-nitrosylated molecules and caspase-3 colocalize in cardiomyocytes treated with DOX and SNAP (Fig. 7C). However, very little fluorescence signal was generated with an antibody against S-nitrosocysteine in cardiomyocytes treated with DOX only. Cotreatment with HgCl2 and SNAP in DOXtreated cardiomyocytes also generated little fluorescence signal. These results provide further support for the hypothesis that the inhibition of caspase-3 activity by S-nitrosylation is one of the major mechanisms by which NO inhibits apoptosis.
Fig. 6. A. Caspase-3 activity (pmol/µl) in lysates from DOX-treated cardiomyocytes. The inhibitory effect of SNAP on caspase-3 activity is reversed by incubation with HgCl2 (100 µmol/l) for 24 h (solid triangles). B. Changes in protein level of cleaved caspase-3 and phospho-JNK in cardiomyocytes treated with DOX evaluated by immunoblot analysis. The mean ± S.D. is shown. #P < 0.01 compared to DOX-only samples. †P < 0.01 compared to cells treated with DOX + SNAP.
inhibited the activation of caspase-3 in DOX-treated cardiomyocytes, we next examined whether S-nitrosylation was involved in the activation of caspase-3. Cotreatment with HgCl2 and SNAP of DOX-treated cardiomyocytes upregulated caspase-3 activity significantly by 1.9-fold compared with treatment with SNAP alone (Fig. 6A). Then, to examine whether NO also affects other apoptotic signal transductions, we examined the effect of NO for the phosphorylation of JNK. As shown in Fig. 6B, DOX activates phospho-JNK irrespective of HgCl2 cotreatment. On the other, cleaved caspase3 protein level was decreased by treatment with SNAP and reverted partially by cotreatment with HgCl2. These results indicate that HgCl2 cleaved S-nitrosothiols and thereby reduced the inhibitory effect of SNAP on caspase-3 activity. 3.6. Role of S-nitrosylation of caspase-3 in the anti-apoptotic effect of NO To clarify whether caspase-3 is nitrosylated by NO in cardiomyocytes, we did three experiments to examine
4. Discussion This study is the first to show that NO inhibits apoptosis in cardiomyocytes by S-nitrosylation of caspase-3. This conclusion was based on the findings that the NO donor, SNAP, was able to inhibit cardiomyocyte apoptosis induced by DOX and reduced caspase-3 activity; DOX-induced apoptosis was reversed by denitrosylation with addition of HgCl2; and immunoprecipitated caspase-3 in cardiomyocytes treated with the SNAP was heavily nitrosylated. The anti-apoptotic effects of NO in cardiomyocytes have been classified as cGMP-dependent and -independent. NO activates cGMP signaling through interaction with the heme group of guanylate cyclase. The production of cGMP causes activation of cGK that leads to and reveals the protective effect [24]. The anti-apoptotic effects of NO can also be mediated through a number of mechanisms independent of cGMP signaling such as activation of p38 MAPK [25], PI3-K/Akt [26], reduction of mitochondrial Ca2+ uptake [27], and a decrease in cyclin A-associated kinase activity [28]. We confirmed that cGK activity does not mediate the inhibitory effect of NO on caspase-3 activity and S-nitrosylation of caspase-3 by NO is a novel cGMP-independent anti-apoptotic mechanism in cardiomyocytes. S-nitrosylation is a ubiquitous post-translational protein modification with broad regulatory purview, analogous in that
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Fig. 7. A. NO content in immunoprecipitated caspase-3 from cardiomyocytes measured by Saville-Griess assay (*P < 0.01 vs. SNAP (–) group). B. S-nitrosylation of immunoprecipitated caspase-3 (*P < 0.01 vs. SNAP (–) group). C. Immunofluorescent staining for caspase-3 and S-nitrosylated protein observed with a laser scanning confocal microscope. Caspase-3 protein is highly expressed in DOX-treated cardiomyocytes (rhodamine, red), and co-expression of S-nitrosylated caspase-3 (FITC, green) is increased in cardiomyocytes treated with SNAP. Representative results are shown. All photographs were taken at the same magnification (100×).
respect to protein phosphorylation. However, the analysis of S-nitrosylated protein in situ has been impeded by substantial technical barriers, and there is little direct evidence for cellular protein S-nitrosylation. Recently, Jaffrey et al. [17] developed a new methodology that lowered the technical hurdles and significantly increased experimental access to NO biology. By using this technique, we confirmed directly that NO inhibits apoptosis by preventing the activation of caspase3 via S-nitrosylation. Pro-apoptotic caspase-3 consists of two subunits, p12 and p17, that form a heterodimer to build the active protease. Subunit p17 contains a reactive cysteine at position 163 in the
catalytic center that is essential for enzyme activity. NO inhibits the p17 subunit via S-nitrosylation of this critical cysteine [2]. Thus, the increased S-nitrosylation of the p17 subunit after exposure to NO donors contributes to the anti-apoptotic effect of NO in cardiomyocytes. Recent studies have found that growth factors such as insulin-like growth factor I, hepatocyte growth factor, and erythropoietin have a protective effect on cardiomyocytes in the failing heart [29–31]. Some investigators have suggested that these growth factors enhance endogenous NO production by various cells [32–34]. Exogenous NO production by non-myocytes surrounding cardiomyocytes, such as endothe-
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lial cells, macrophages, and smooth muscle cells, is an important mechanism regulating apoptosis in cardiomyocytes. Furthermore, some studies have found that various pharmacological interventions can restore NO synthesis and inhibit downstream caspase-3 activation in failing human myocardium [35,36]. Taken together, the protective effect of these growth factors may be due partially to enhancement of NO production in cells surrounding cardiomyocytes. The results of our study show these growth factors to be attractive as possible therapeutic agents for preventing apoptosis. Recent reports suggested that p21ras and thioredoxin (Trx) are also nitrosylated and work as anti-apoptotic molecules in endothelial cells [37,38]. An increased S-nitrosylation of p21ras enhances its activation and contributes to activation of MAP kinase signaling that may regulate apoptosis. Trx can exert its anti-apoptotic activity via redox regulatory activity by S-nitrosylation at cysteine 69. Schonhoff et al. [39] reported that nitrosylated cytochrome c released from mitochondria has a proapoptotic effect, but is unknown how this molecule works in cardiomyocytes. Therefore, it is worth to investigate the intra-cellular reaction of S-nitrosylation in cardiomyocyte, especially in apoptosis. It is known that the effects of NO on apoptosis are not only inhibitory as shown in this study but also stimulatory [40]. It has been the subject of discussion as to why the same molecule should perform both these opposite tasks and it is thought that the biological conditions, such as the redox state, concentration, exposure time and the combination with oxygen, superoxide and other molecules, determine the net effects of NO on apoptosis [41]. In this experimental condition, these factors to the cells may affect S-nitrosylation of caspase-3. Also, NO is implicated in both apoptotic and necrotic cell death depending on the NO chemistry and the cellular biological redox state [41]. DOX is known to generate oxygenderived free radicals, and NO and oxygen-derived free radicals produce peroxynitrite, which may induce cellular damage. In the present study, we showed that high-dose SNAP induces apoptosis and also necrosis in DOX-treated cardiomyocytes, and peroxynitrite production significantly increased after stimulation with SNAP in dose-dependent manner. Taken together, a large quantity of peroxynitrite formed by the combination of DOX and high-dose NO may play a detrimental role for cardiomyocytes. In conclusion, exogenous NO induces S-nitrosylation of caspase-3, blocks its activation, and thereby partially inhibits DOX-induced apoptosis in cardiomyocytes. The ability of NO to inhibit downstream caspase-3 means that NO may be able to rescue cardiomyocytes from apoptosis even after the caspase cascade has been activated. Depletion of NO from non-myocytes or cardiomyocytes increases caspase-3 activity and might be associated with progressive ventricular remodeling and heart failure. The bioavailability of NO production might inhibit caspase-3 activation and prevent cardiomyocyte loss and remodeling in heart failure. Although, cardiomyocytes occupy over 75% of the structural space of the
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heart, the remaining non-myocytes consist cardiac fibroblasts, endothelial cells, coronary smooth muscle cells, and so on. To confirm whether supplementation of NO is beneficial for DOX-induced heart failure, further study must await, such as the experiments using non-myocytes and in vivo study.
Acknowledgements This study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan; a Research Grant for Diseases from the Ministry of Health and Welfare of Japan; and a Grant from the Japan Cardiovascular Research Foundation.
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