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Cardiovascular Research 2003 59(2):308-320; doi:10.1016/S0008-6363(03)00425-5
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Copyright © 2003, European Society of Cardiology

Nitric oxide inhibits ischemia/reperfusion-induced myocardial apoptosis by modulating cyclin A-associated kinase activity

Yasuhiro Maejimaa, Susumu Adachia,*, Hiroshi Itoa, Kiyoshi Noboria, Mimi Tamamori-Adachib and Mitsuaki Isobea

aDepartment of Cardiovascular Medicine, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan
bDepartment of Biochemical Genetics, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo, Tokyo 113-8519, Japan

* Corresponding author. Tel.: +81-3-5803-5218; fax: +81-3-5803-0133. sadachi.cvm{at}tmd.ac.jp

Received 25 December 2002; accepted 22 April 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: Ischemia/reperfusion in the heart causes myocardial apoptosis and increase nitric oxide (NO) production. We have reported that myocardial apoptosis is related to activation of cell cycle regulatory proteins. However, the role of nitric oxide (NO) in ischemia/reperfusion-induced apoptosis is still unclear. This study was designated to elucidate novel apoptosis mechanisms induced by ischemia/reperfusion, especially the interaction between NO and cell cycle regulators. Methods and results: Neonatal cardiomyocytes from 1- or 2-day-old Wistar rats were subjected to 1-h ischemia and then to reperfusion. The rate of cardiomyocyte apoptosis increased significantly after 24 h of reperfusion as evaluated by TUNEL analysis. NO increased 1.8-fold after 15 min of reperfusion in cardiomyocytes. After 36 h of reperfusion, the apoptosis rate was greatly increased by the NO synthetase inhibitor, Nitro-L-arginine methyl ester (L-NAME), and decreased by the NO donor of S-nitroso-N-acetylpenicillamine (SNAP). Immunoblot analysis showed that the protein levels of cyclin A accumulated in a time-dependent manner in response to ischemia/reperfusion, and L-NAME inhibited this response. Ischemia/reperfusion also increased the activity of cyclin A-associated kinase, and the apoptosis was inhibited by infection of dominant-negative cdk2 adenovirus. To clarify the involvement of p21cip1/waf1 protein, which is the suppressor of cyclin A-associated kinase, we performed immunoblot analysis and examined its kinase activity. Treatment of cardiomyocytes with L-NAME suppressed the p21cip1/waf1 protein level and increased the cyclin A-associated kinase activity. The addition of SNAP showed inverse results. Conclusion: Our data indicates that NO released from cardiomyocytes under condition of ischemia/reperfusion exerts an antiapoptotic effect by modulating cyclin A-associated kinase activity via p21cip1/waf1 accumulation.

KEYWORDS Apoptosis; Ischemia; Myocytes; Nitric oxide; Reperfusion

This article is referred to in the Editorial by C. Pignatti and C. Stefanelli (pages 268–270) in this issue.


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Nitric oxide (NO) is an endogenous regulatory molecule involved in a variety of physiological processes. These include a broad range of activities such as regulation of vessel tone, proliferation of smooth muscle cells, and cytoprotection from ischemic injury [1]. NO is generated from L-arginine via NO synthetases and released from endothelial cells [2] as well as cardiomyocytes [3,4]. Recent investigations showed that NO plays an important role in protecting myocardium from ischemia/reperfusion injury [5–9].

Although prompt reperfusion within a narrow time window has significantly reduced early mortality from acute myocardial infarction, post-infarction heart failure resulting from ventricular remodeling is reaching epidemic proportions. During therapeutic intervention of acute myocardial infarction, the heart is still partially injured by reperfusion following ischemia. The ischemic episode causes cellular damage and cell loss attributable to apoptosis of cardiomyocytes [10]. If expansion of the apoptotic regions is to be prevented, the underlying mechanisms have to be more thoroughly investigated.

The induction of apoptosis by various stimuli involves mitochondria, death receptor, MAP/JNK, and PI-3K/Akt [11–14]. Moreover, apoptosis of proliferating cells involves cell cycle regulators [15–17]. The cardiomyocyte is terminally differentiated and irreversibly withdrawn from the cell cycle. However, the cardiomyocytes express some cell cycle regulators. Recently, we and others showed that certain apoptotic mechanisms modulate cell cycle regulators. For example, cyclin A-associated kinase activity is upregulated by an apoptotic mechanism; inhibition of cyclin A-associated kinase activity suppresses apoptosis [18–20].

Recent studies indicate that NO modulates the expression of cell cycle regulatory proteins and cyclin-dependent kinase activities [21–23]. In the heart, NO formation is elevated after ischemia/reperfusion [24], suggesting that NO may be involved in inhibition of myocardial apoptosis via modulation of cell cycle regulators.

In the present study, we have examined the interaction between NO and cell-cycle regulatory proteins during ischemia/reperfusion-induced apoptosis in neonatal rat cultured cardiomyocytes.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
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 [25]. Cardiac fibroblasts were also prepared [26]. The cardiomyocytes were incubated in Eagle's minimum essential medium (MEM) (Sigma, St. Louis, MO, USA) supplemented with 5% calf serum (CS) (JRH Biosciences, Lenexa, KS, USA) at 37°C. After the growth medium was changed, cells were exposed to ischemia/reperfusion condition. To obtain ischemia/reperfusion stimuli, cells were exposed for 1 h to severe ischemic conditions in a Plexiglass chamber containing serum-free medium purged with a constant stream of water-saturated 95% N2/5% CO2. The cells were subsequently under normoxic conditions with 5% CS medium for reperfusion. Zero time point was assigned the beginning of the reperfusion step. All experiments were performed 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 Apoptotic assays
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 (Sysmex cell counter CDA-500, Long Grove, IL, USA) [27,28]. For in situ TUNEL assay, floating cells were recovered from the medium by centrifugation and pooled with the adherent cells collected by trypsinization. Cells were spun down onto poly-L-lysine-coated slides, and TUNEL assays were performed with a commercially available kit for detecting end-labeled DNA according to the manufacturer's instructions (ApopTag, Oncor, Gaithersburg, MD, USA) and with anti-digoxigenin-rhodamine (Roche Diagnostics, Indianapolis, IN, USA) [29]. 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 [30]. This method is based on the observation that cells undergoing apoptosis when fixed in ethanol and stained with propidium iodide (PI) (Sigma) 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, USA). 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 NO production
NO production after ischemia/reperfusion in cardiomyocytes was measured with phenol red-free MEM (Invitrogen, San Diego, CA, USA). NO was measured as nitrite [31] by Griess method according to the kit manufacturer's protocol (Dojindo, Kumamoto, Japan). Briefly, after nitrate was reduced to nitrite, nitrite was mixed with the Griess’ reagent (1% sulfanilamide, 0.1% naphthylethylene diamine hydrochloride, and 2.5% phosphoric acid), and absorbance of the reaction product was read at 540 nm after a 10-min incubation. Standard curves were prepared with known concentrations (5–50 µmol/l) of NaNO2. Nitric oxide synthetase (NOS) was inhibited with nitro-L-arginine methylester (L-NAME, Sigma) in the experiments described below. To evaluate the effect of NO donor, S-nitroso-N-acetylpenicillamine (SNAP, BIOMOL Research Laboratories, Plymouth Meeting, PA) was used.

2.5 Antibodies and immunoblotting
The following antibodies and reagents were used: polyclonal antibodies for rabbit cyclin A (sc-751), cyclin B1 (sc-595), cyclin E (sc-481), cdk2 (sc-163), cdk4 (sc-260), and p21cip1/waf1 (sc-397), monoclonal antibody for mouse cyclin D1 (sc-450) (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and {alpha}-sarcomeric actin (M0874, DAKO. Glostrup, Denmark). Electrophoresis was performed on 10 or 15% SDS–polyacrylamide gels. Sample volumes were adjusted by gel staining with Coomassie brilliant blue. Gels were transferred to nitrocellulose membranes by semi-dry electrotransfer and immunoblotting was performed as previously described [32]. All experiments evaluated densitometric analysis by NIH image software (National Institutes of Health, Bethesda, MD).

2.6 Adenoviral infection of dominant-negative cdk2 and treatment of chemical inhibitor of cdk2
The recombinant adenoviruses of dominant-negative cdk2 (dncdk2) were prepared as described previously [18]. Cardiomyocytes were infected by adenovirus at the 50 multiplicity of infection (moi) and incubated for 1 h with brief agitation every 15 min. After infection, the medium was replaced by culture medium. Cdk2 activity was also reduced by adding Roscovitine (100 µmol/l, Calbiochem, La Jolla, CA, USA) to the medium.

2.7 In vitro histone H1 kinase assay
The cardiomyocytes were harvested at various time points after ischemia/reperfusion. Whole-cell extracts (100 µg) were precleaned with protein A/G agarose beads (sc-2003, Santa Cruz Biotechnology), and immunoprecipitations were performed with anti-cdk2, anti-cyclin E, or anti-cyclin A polyclonal antibody overnight at 4°C. After the pellets were washed twice in lysis buffer (50 mmol/l Hepes (pH 7.9), 150 mmol/l NaCl, 0.1 mmol/l EDTA, 0.1 mmol/l EGTA, 0.1% NP-40, 0.4 mmol/l NaF, 0.4 mmol/l Na3VO4, 10% glycerol, 0.1 mmol/l PMSF, 1 mmol/l DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A) and twice in kinase buffer (50 mmol/l Hepes (pH 8.0), 10 mmol/l MgCl2, 2.5 mmol/l EGTA, 10 mmol/l β-glycerophosphate, 1 mmol/l NaF, 0.1 mmol/l Na3VO4, 0.1 mmol/l PMSF, 1 mmol/l DTT, 10 µg/ml aprotinin, 10 µg/ml leupeptin, and 10 µg/ml pepstatin A), they were incubated in 25 µl of kinase assay solution (2 µg histone H1, 10 µmol/l ATP, 4 µCi [{gamma}-32P]ATP in kinase buffer) for 20 min at 30°C. The mixtures were boiled for 3 min, loaded onto 10% SDS–polyacrylamide gels, and exposed to X-ray film (Hyperfilm MP, Life Science Amersham Biosciences, Buckinghamshire, UK) after electrophoresis.

2.8 Statistical analysis
Data are shown as mean±S.D. values. Differences were analyzed with one-way analysis of variance (ANOVA) and post-hoc analysis was performed with Bonferroni/Dunn test. A P value of less than 0.01 considered to indicate statistical significance.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Ischemia/reperfusion induced apoptosis in cardiomyocytes
Apoptosis in response to ischemia/reperfusion injury was investigated in primary neonatal rat cardiomyocytes. The increased end-labeling of DNA in the ischemia/reperfusion-treated cardiomyocytes as detected by the TUNEL method is shown in Fig. 1A. There were significant increases in the number of end-labeled cardiomyocytes evaluated after 24 h of ischemia/reperfusion (control, 9.3±0.8% vs. ischemia/reperfusion, 20.6±2.7%). A decreased number of viable cells among the ischemia/reperfusion-treated cardiomyocytes as determined by cell counts (control, 96.8±0.1% vs. ischemia/reperfusion, 87.9±0.2%) is shown in Fig. 1B.


Figure 1
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  		Fig. 1 Evaluation of ischemia/reperfusion-induced apoptosis in cardiomyocytes. (A) Quantitative analysis of apoptosis induced by ischemia/reperfusion. Percentages of apoptotic cells were calculated by TUNEL staining. (B) Quantitative analysis of cellular viability during ischemia/reperfusion by cell counter. Values were calculated from three independent experiments, and mean±S.D. values are shown. * vs. time 0 (P<0.01). (C) Representative data of flowcytometric analysis to detect sub G1 phase in cardiomyocytes.

 
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 cells at the subG1 phase increased after 24 h of ischemia/reperfusion. These results demonstrate that ischemia/reperfusion significantly accelerates apoptosis in cardiomyocytes.

3.2 NO production and effect of NOS inhibitor in ischemia/reperfusion induced apoptosis
NO concentration was measured in culture medium under ischemia/reperfusion conditions. NO production was 1.8-fold higher in cardiomyocytes after 15 min of ischemia/reperfusion as compared to cardiomyocytes under normoxic (control) conditions. Moreover, L-NAME (1 mmol/l) inhibited ischemia/reperfusion-induced NO production (Fig. 2A, upper panel). Unlike cardiomyocytes subjected to ischemia/reperfusion, there was no increase in NO production of cardiomyocytes by readdition of serum in the medium under normoxic condition. Cardiomyocytes conditions, either under hypoxia with or without serum, showed no increase in NO production. Furthermore, for testing whether NO was the effector for apoptosis in this model, cardiomyocytes were exposed to L-NAME and SNAP (1 mmol/l) prior to 24 h of ischemia/reperfusion. Thirty-six-hour ischemia/reperfusion-induced apoptosis as evaluated by TUNEL assay was significantly higher in cardiomyocytes treated with L-NAME than in cardiomyocytes receiving no L-NAME. On the other hand, the apoptosis rate of cardiomyocytes treated with SNAP was significantly lower than in cardiomyocytes without SNAP under ischemia/reperfusion [ischemia/reperfusion only: 23±1.4%, L-NAME (+): 35.7±0.9%, SNAP (+): 18.7±1.4%].


Figure 2
Figure 2
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  		Fig. 2 (A) NO production and the effect of NOS inhibitor L-NAME (1 mmol/l) in ischemia/reperfusion-induced apoptosis. Upper panel: time course of nitrite concentration in the medium of cultured cardiomyocytes with or without L-NAME after ischemia/reperfusion, and in normoxic condition with or without serum in the medium. Lower panel: time course of nitrite concentration in the media of cultured cardiomyocytes under several conditions. NO was measured as nitrite by the Griess’ method. Values were calculated from three independent experiments, and as mean±S.D. values are shown. * vs. time 0 (P<0.01). (B) Quantitative analysis of apoptosis during ischemia/reperfusion in control, L-NAME-treated and SNAP-treated cardiomyocytes by TUNEL staining. (C) Quantitative cell counter analysis of cellular viability during ischemia/reperfusion in control, L-NAME-treated and SNAP-treated cardiomyocytes. Values were calculated from three independent experiments, and mean±S.D. values are shown. * vs. control samples (P<0.01). ** vs. non-treatment samples (P<0.01). (D) Representative data of hypodiploid DNA content analysis by flowcytometry during ischemia/reperfusion.

 
3.3 Change of cell-cycle regulator proteins in ischemia/reperfusion-induced apoptosis and the effect of endogenous NO in cardiomyocytes
To determine whether the protein levels of cell-cycle regulators change in ischemia/reperfusion, we used immunoblotting to examine several cell-cycle regulators, ie cdks and cyclins. Under ischemia/reperfusion conditions, cyclin B, cyclin D, cyclin E, cdk2 and cdk4 protein did not change significantly, whereas the level of cyclin A gradually increased in a time-dependent manner (Fig. 3A,C). A comparison of cyclin A expression in cardiomyocytes and apoptosis-resistant cardiac fibroblast is shown in Fig. 3B. The level of cyclin A protein did not change in cardiac fibroblasts. These results suggest that cyclin A expression is related to apoptosis in cardiomyocytes.


Figure 3
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  		Fig. 3 Changes in cell-cycle-related proteins in cardiomyocytes induced by ischemia/reperfusion evaluated by immunoblot analysis. (A) The protein levels of cyclins A, B, D, E, cdk2, and cdk4. (B) Comparison of cyclin A protein levels between cardiomyocytes and cardiac fibroblasts after ischemia/reperfusion. (C) Densitometric analyses of cell-cycle regulator proteins. Values were calculated from three independent experiments and were normalized to those at time 0. Results are shown as mean±S.D. values. * vs. time 0 (P<0.01).

 
We then examined the effect of L-NAME on cyclin A protein expression under ischemia/reperfusion conditions. The accumulation of cyclin A protein by ischemia/reperfusion was facilitated by L-NAME. Also, we examined the effect of SNAP on cyclin A protein expression under ischemia/reperfusion conditions. The accumulation of cyclin A protein by ischemia/reperfusion was inhibited by SNAP (Fig. 4). This suggests that cyclin A expression is modulated by NO.


Figure 4
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  		Fig. 4 Effects of L-NAME or SNAP on the change in cyclin A protein after ischemia/reperfusion in cardiomyocytes as evaluated by immunoblot analysis. (A) Accumulation of cyclin A protein by ischemia/reperfusion facilitated by L-NAME and decreased by SNAP. (B) Densitometric analyses of cyclin A protein level. Values were calculated from three independent experiments and were normalized to those at time 0. Results are shown as the mean±S.D. values. * vs. time 0 (P<0.01).

 
3.4 Activation of cyclin A-associated kinase activity in response to ischemia/reperfusion-induced apoptosis and the effect of endogenous NO
The function of cyclin A or cyclin E is related to the kinase activity of cdk2 in complex with cyclin A or cyclin E. To confirm that cyclin A-associated or cdk2-associated kinase activity increases during ischemia/reperfusion, cyclin A-associated, cyclin E-associated, and cdk2-associated complexes were immunoprecipitated by cyclin A, cyclin E, and cdk2 antibody, respectively, and assayed for kinase activity in vitro with histone H1 as substrate. We observed a 3.1-fold increase in the phosphorylation of histone H1 in the complex immunoprecipitated by cyclin A and 1.8-fold increase in phosphorylation in the complex immunoprecipitated by cdk2. However, cyclin E-associated kinase activity was hardly changed (Fig. 5). Thus, cyclin A-associated kinase activity is increased under ischemia/reperfusion.


Figure 5
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  		Fig. 5 Effects of L-NAME or SNAP on the change in cyclin A-associated kinase activity in response to ischemia/reperfusion-induced apoptosis in cardiomyocytes. (A) Representative data of histone H1 kinase activity. Cardiomyocytes 24 h after exposure to ischemia/reperfusion in the presence or absence of L-NAME or SNAP were analyzed for cdk2, cyclin A, and cyclin E-associated kinase activity. (B) Densitometric analyses of histone H1 kinase activity. Values were calculated from three independent experiments and were normalized to those at time 0. Results are shown as mean±S.D. values. * vs. control samples (P<0.01). ** vs. non-treatment samples (P<0.01).

 
To examine whether the inhibition of cdk2 activity could also inhibit the ischemia/reperfusion-induced apoptosis, we infected the cells with dncdk2 adenovirus before ischemia/reperfusion or treatment with the chemical cdk2 inhibitor, Roscovitine. When cardiomyocyte were cultured with cdk2 inhibitor, apoptosis was significantly reduced compared with ischemia/reperfusion condition by TUNEL method (Fig. 6).


Figure 6
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  		Fig. 6 (A) Inhibition of apoptosis in cardiomyocytes by dominant-negative cdk2 adenovirus (dncdk2) (50 moi) or Roscovitine (100 µmol/l), chemical cdk2 inhibitor, after 36 h of ischemia/reperfusion. The number of apoptosis evaluated by TUNEL method was expressed as a percentage. Values were calculated from three independent experiments, and mean±S.D. values are shown. * vs. 36 h of ischemia/reperfusion condition (P<0.01). (B) Representative results are shown. Left side: fluorescent (rhodamine) pictures of TUNEL, right side: phase contrast pictures. All photographs were taken at the same magnification (x100).

 
We next examined the effect of L-NAME and SNAP on cyclin A-associated kinase activity. The cyclin A-associated or cdk2-associated kinase activity induced by ischemia/reperfusion was facilitated by the presence of L-NAME. In contrast, these activities were inhibited by SNAP (Fig. 5). Thus, cyclin A-associated kinase activation by ischemia/reperfusion is inhibited by NO.

3.5 Involvement of p21cip1/waf1 activation and effect of endogenous NO on ischemia/reperfusion-induced apoptosis
To understand the mechanism underlying the modulation of cyclin A/cdk2 activity in apoptotic cells, we examined p21cip1/waf1 protein, a cdk2 inhibitor known to play a major role in the regulation of cdk2 activity. Under the ischemia/reperfusion condition, the level of p21cip1/waf1 protein increased gradually in a time-dependent manner as evaluated by immunoblot analysis (5.6±0.5-fold in 24 h). p21cip1/waf1 was significantly lower in cardiomyocytes subjected to ischemia/reperfusion with L-NAME than in cardiomyocytes subjected to ischemia/reperfusion without L-NAME (Fig. 7). Treatment of cardiomyocytes with SNAP in ischemia/reperfusion condition caused significant p21cip1/waf1 protein accumulation as compared to cardiomyocytes subjected to ischemia/reperfusion without SNAP. These results suggest that NO is an upstream activator of p21cip1/waf1.


Figure 7
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  		Fig. 7 Changes in p21cip1/waf1 protein in cardiomyocytes induced by ischemia/reperfusion evaluated by immunoblot analysis. (A) Time course of p21cip1/waf1 protein after ischemia/reperfusion. (B) Accumulation of p21cip1/waf1 protein by ischemia/reperfusion decreased by L-NAME and facilitated by SNAP. (C) Densitometric analyses of p21cip1/waf1 protein level. Values were calculated from three independent experiments and were normalized to those at time 0. Results are shown as mean±S.D. values. * vs. control samples (P<0.01). ** vs. non-treatment samples (P<0.01).

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
It is well documented that NO is a regulator of cell physiology and plays an important role in cytoprotection [1]. Nonetheless, the cytoprotective function of NO in cardiomyocytes is still not precisely understood. In the present study, we have shown that ischemia/reperfusion induces apoptosis and increases the production of NO in cultured cardiomyocytes. NO acts to inhibit apoptosis mediated by the inhibition of cyclin A/cdk2 activity associated with upregulation of cdk inhibitor p21cip1/waf1. Our study is the first to show that NO plays a novel role in protecting cardiomyocytes from apoptosis by modulating cell cycle regulator proteins.

Ischemia/reperfusion occurs in the ischemic region produced by angina pectoris or myocardial infarction and is often associated with arrhythmia, inflammation, and myocardial dysfunction. This in vitro experimental approach with cultured cardiomyocytes allows evaluation of the direct effect of ischemia/reperfusion on cardiomyocytes in the absence of additional effects induced by inflammatory cells. The present study showed that ischemia/reperfusion induced apoptosis in cardiomyocytes as evaluated by flowcytometric analysis, number of viable cells, and TUNEL assay. Moreover, endogenous NO synthesis in cardiomyocytes was increased under conditions of ischemia/reperfusion [24,33]. Compared to that of the continuous hypoxic condition with or without serum, significantly increased NO production was detected in relation to ischemia/reperfusion stimuli. This suggests that ischemia/reperfusion is less damaging than ischemia alone.

Apoptosis, which is sometimes occurred in uncontrolled condition of cell proliferation, implicates in a wide variety of physiological and pathological processes [27]. In recent reports, cdk activity was shown to be involved in apoptosis. For instance, apoptosis induced by deprivation of growth factor in human endothelial cells is associated with an upregulation of cyclin A-associated cdk2 activity, and inhibition of this activity suppresses apoptosis [15]. Moreover, cdks act at an early step in the pathway of KCL withdrawal-induced apoptotic death of cerebellar granule cells, which cells are terminally differentiated and similar to cardiomyocytes [17]. In cardiomyocytes, cell cycle regulator proteins may play physiologically and pathophysiologically different roles. We previously showed that myocardial apoptosis is related to activation of cyclin A-associated kinase under hypoxic conditions [18]. Our results provide new insight into the understanding of cell cycle regulators and apoptosis in cardiomyocytes. Here we have shown that cyclin A protein and cyclin A-associated kinase activity increased concomitantly with apoptosis in cardiomyocytes after ischemia/reperfusion. Further, the intensities of apoptosis and cyclin A-associated kinase activity were enhanced by L-NAME and were decreased by SNAP, suggesting that myocardial apoptosis in response to ischemia/reperfusion is less damaging by endogenous NO, which modulates cyclin A-associated kinase activity (Fig. 8). Several investigators reported that NO in proliferative cells inhibits cell proliferation by suppressing cyclin A-associated kinase activity through upregulation of p21cip1/waf1 protein [21,23,34]. We and several other groups reported that caspase-3, an activator of apoptosis, truncates p21cip1/waf1 cdk inhibitorand induces cdk activity in apoptotic cells [15,18,35]. Thus, NO is likely to increase the protein level of p21cip1/waf1, leading to suppression of cdk activity. In the present study, we showed that p21cip1/waf1 protein increased in ischemia/reperfusion-induced apoptosis in cardiomyocytes, and this change was suppressed by L-NAME and was facilitated by SNAP, suggesting that NO attenuates apoptosis through the upregulation of p21cip1/waf1 protein. In a previous study, NO was shown to function as a potential regulator of the cell cycle involving the induction of p21cip1/waf1 in aortic fibroblasts through a cGMP mediated transcriptional mechanism [34]. Moreover, several studies have shown that the underlying mechanism of p21cip1/waf1 upregulation by NO to be enhanced expression of p21cip1/waf1 protein [23] or prevention of p21cip1/waf1 protein degradation via an ubiquitin-proteasome pathway [36]. In cardiomyocytes, the ubiquitination of p21cip1/waf1 might be activated in ischemia/reperfusion. The effects of NO on apoptosis are not only inhibitory as shown in this study but also stimulatory [37]. It is thought that a complex set of factors, such as the type and timing of an apoptotic stimulus, the source of NO and the redox chemistry within cells, will determine the net effects of NO on apoptosis. These factors to the cells may affect c-GMP response and/or S-nitrosylation for several apoptosis regulatory pathways [38].


Figure 8
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  		Fig. 8 Schematic illustration of the regulation of ischemia/reperfusion-induced myocardial apoptosis by endogenous nitric oxide. We provide data showing that nitric oxide produced by cardiomyocytes stimulated by ischemia/reperfusion both decreases cyclin A protein level and increases p21cip1/waf1 protein level. This may lead to the inhibition of cyclin A-associated kinase activity.

 
In conclusion, we showed that ischemia/reperfusion promotes apoptosis in cardiomyocytes in vitro by modulating the activity of novel cell cycle regulators and enhancement of endogenous NO production by ischemia/reperfusion attenuates against apoptosis by inhibiting cdk activity via p21cip1/waf1. This might represent a physiological mechanism by which ischemia/reperfusion causes cardiac apoptosis and cytoprotection simultaneously. The present study sheds light on the molecular mechanism underlying cardiac apoptosis and may point to a novel therapeutic target for controlling cardiac apoptosis induced by ischemia/reperfusion.

Time for primary review 22 days.


   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 Japan Cardiovascular Research Foundation.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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