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Cardiovascular Research 2000 45(3):671-678; doi:10.1016/S0008-6363(99)00347-8
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Copyright © 2000, European Society of Cardiology

Inhibition of endogenous nitric oxide synthase potentiates ischemia–reperfusion-induced myocardial apoptosis via a caspase-3 dependent pathway

Ulrike Weilanda, Judith Haendelera, Christian Ihlingb, Udo Albusc, Wolfgang Scholzc, Hartmut Ruettenc, Andreas M. Zeihera and Stefanie Dimmelera,*

aMolecular Cardiology, Department of Internal Medicine IV, University of Frankfurt, Theodor-Stern-Kai 7, 60596 Frankfurt/M, Germany
bInstitut of Pathologie, University of Freiburg, Freiburg, Germany
cHoechst AG, Cardiovascular Research H821, Frankfurt/M, Germany

* Corresponding author. Tel.: +49-69-6301-7440; fax: +49-69-6301-7113 Dimmeler{at}em.uni-frankfurt.de

Received 1 June 1999; accepted 27 August 1999


   Abstract
Top
 Abstract
1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Objective: Apoptosis of cardiomyocytes may contribute to ischemia–reperfusion injury. The role of nitric oxide (NO) in apoptosis is controversial. Therefore, we investigated the effect of NO synthase inhibition on apoptosis of cardiomyocytes during ischemia and reperfusion and elucidated the underlying mechanisms. Methods and results: Isolated perfused rat hearts (n=6/group) were subjected to ischemia (30 min) and reperfusion (30 min) in the presence or absence of the NO synthase inhibitor NG-mono-methyl-L-arginine. Reperfusion induced cardiomyocyte apoptosis as assessed by immunohistochemistry (TUNEL-staining) and the demonstration of the typical DNA laddering. Apoptosis during reperfusion was associated with the cleavage of caspase-3, the final down-stream executioner caspase, whereas the protein levels of the anti-apoptotic protein Bcl-2 and the pro-apoptotic protein Bax were unchanged. Inhibition of the NO synthase drastically increased ischemia and reperfusion-induced apoptosis of cardiomyocytes. Moreover, the NO synthase inhibitor enhanced the activation of caspase-3, suggesting that NO interferes with the activation of caspases in ischemia–reperfusion. Conclusion: The results of the present study demonstrate that inhibition of endogenous NO synthesis during ischemia and reperfusion leads to an enhanced induction of apoptosis, suggesting that the endogenous NO synthesis protects against apoptotic cell death. Inhibition of NO synthesis thereby activates the caspase cascade, whereas the Bcl-2/Bax protein levels remained unchanged.

KEYWORDS Apoptosis; Ischemia; Nitric oxide; Reperfusion


   1 Introduction
Top
 Abstract
 1 Introduction
2 Methods
 3 Results
 4 Discussion
 Acknowledgments
 References
 
Myocardial cell death has been shown to contribute to ischemia–reperfusion injury. Cell death may be differentiated into two different kinds: necrosis and apoptosis. Necrotic cell death is associated with membrane permeability and lysis of the cells [1]. In contrast, apoptosis results in single cell loss with characteristic cell shrinkage, membrane-blebbing, DNA-fragmentation and chromatin-condensation but no increase in plasma membrane permeability [2,3]. Apoptotic cell death of cardiomyocytes was observed in heart failure [4] as well as in reperfusion injury [5–7]. However, the mechanisms underlying ischemia–reperfusion-induced apoptosis are poorly understood. Apoptosis is regulated through different signaling pathways, which are composed of the highly conserved cell death genes of the nematode C. elegans, Ced-3, Ced-4 and Ced-9. Mammalian apoptosis is mainly regulated by caspases, the homologue to Ced-3, Apaf-1 (homologue to Ced-4) and the Bcl-2 family (homologues to Ced-9) [8–10]. The bcl-2 family consists of different apoptosis promoting (e.g. Bax) as well as apoptosis inhibiting (e.g. Bcl-2 and Bcl-xL) members. These members are able to form homo- and heterodimers [10,11]. The ratio between anti-apoptotic and pro-apoptotic Bcl-2-family members seems to be critical for cell survival (for review see [11]). The pro-apoptotic cysteine protease family of caspases, the homologues of ced-3, are activated by a proteolytic cascade where inactive caspase precursors are cleaved to a larger and a shorter catalytic subunit [9,12]. The cleavage of caspase-3 into p17 and p12 plays an important role as the final common apoptosis executioner [13], which activates the caspase-activated DNase [14].

Nitric oxide (NO) is an important physiological mediator, which plays a major role in vascular biology [15] and heart failure [16]. Moreover, NO exerts cytotoxic effects [17] and has been shown to trigger apoptotic cell death in various cell types including cardiac myocytes [18–20]. Controversially, anti-apoptotic effects of NO have also been reported in vitro [21–24]. The role of endogenous NO in apoptosis caused by ischemia–reperfusion is not known.

Therefore, the aim of the present study was to investigate the contribution of endogenous NO to ischemia- and reperfusion-induced apoptosis in isolated perfused rat hearts. Furthermore, we examined the contribution of caspases and the Bcl-2-like proteins Bcl-2 and Bax in regulation apoptosis of cardiac myocytes. Since we and others previously demonstrated that NO inhibits caspase activity [21,25,26], we further investigated the effect of the NO synthase (NOS) inhibitor on caspase activation.


   2 Methods
Top
 Abstract
 1 Introduction
 2 Methods
3 Results
 4 Discussion
 Acknowledgments
 References
 
2.1 Reagents
35S-Methionine, ([32P]dCTP, horseradish peroxidase-conjugated secondary antibodies and ECL-reagents were purchased from Amersham (Braunschweig, Germany). Rabbit polyclonal antibodies against Bcl-2, Bax and p17 were obtained from Santa Cruz (Heidelberg, Germany). The T7 in vitro transcription–translation system was from Promega (Madison, USA).

2.2 Animal model
Animal experiments were performed according to the National Institute of Health Guidelines for the use of experimental animals and were approved by the local Ethical Committee for Animal Protection. Male Wistar rats (450–500 g body weight) were anaesthetised with pentobarbital (60 mg/kg i.p.) and subsequently heparinized (500 I.U./100 g BW i.p.). Once stable anaesthesia was achieved, the chest was opened and the hearts were quickly removed and immersed in physiological cold buffer (4°C) to produce immediate cessation of contractility. Hearts (average weight of 1.8 g) were perfused in a Langendorff apparatus with modified Krebs–Henseleit buffer (113.8 mM NaCl, 22 mM NaHCO3, 4.7 mM KCl, 1.2 mM KH2PO4, 1.1 mM MgSO4, 2.5 mM CaCl2, 11 mM glucose, 2 mM Na-pyruvate) at 37°C. After an initial perfusion of 30 min, stop flow global ischemia was induced for 30 min (ischemia, n=6). For ischemia–reperfusion, ischemic hearts (30 min ischemia) were reperfused for additional 30 min (reperfusion, n=6). Endogenous NO synthesis was inhibited by 1 mM L-NG-mono-methyl-arginine (L-NMMA, Alexis, Grünberg, Germany). Therefore, L-NMMA was added to the perfusate 10 min prior to stop flow global ischemia (30 min) (ischemia+L-NMMA, n=6). To test the effect of L-NMMA on ischemia–reperfusion, L-NMMA was infused 10 min prior to ischemia (30 min) and during reperfusion for 30 min (reperfusion+L-NMMA, n=6). As a control L-NMMA was continuously infused for 70 min without ischemia (L-NMMA, n=3). The hearts of the sham group were obtained after continuous perfusion for 70 min (control, n=6). After the experiments, hearts were divided and either rapidly frozen in liquid nitrogen and stored at –80°C for DNA and protein isolation or fixed in 4% buffered formaldehyde and then embedded in paraffin for immunohistochemical analyses.

2.3 Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL)
The detection of DNA strand breaks in situ by TUNEL was performed as described by Gavrieli et al. [27] with minor modifications [28]. In brief, 5 µM sections were incubated with 3% citric acid for 1 h and subsequently with proteinase K 20 µg/ml for 15 min at room temperature. After the quenching of endogenous peroxidase, sections were rinsed in terminal deoxynucleotidyl transferase–buffer (30 mM Tris, 140 mM sodium cacodylate, 1 mM cobalt chloride) at pH 7.2 and incubated with 0.3 U/µl terminal deoxynucleotidyl transferase (Sigma, Munich, Germany) and biotinylated-dUTP (1:200, Boehringer, Mannheim, Germany) in TdT buffer for 60 min at 37°C. Labelled nuclei were detected with Vectastain ABC (Vector Labs, Grünberg, Germany) and peroxidase activity was visualised by 3-amino-9-ethylcarbazole (AEC) to yield a brown reaction product. As a positive control, tissue of follicular hyperplasia of the appendix was used and gave the expected positive staining by TUNEL of tingible bodies in the germinal centers. For quantitative analysis, TUNEL-positive cells in five different sectors with approximately 500 cells/sector were counted (i.e. 2500 cells/animal).

2.4 DNA-laddering
DNA was extracted by phenol–chloroform extraction as described previously [29]. The DNA samples were treated with 5 U of Klenow polymerase using 0.5 µCi of [{alpha}-32P]-dCTP in the presence of 10 mM Tris–HCl (pH 7.5) and 5 mM MgCl2. The reaction was terminated after addition of 10 mM EDTA and the unincorporated nucleotides were removed by Sephadex G-50 columns. The radioactive labelled DNA was separated by gel electrophoresis (1.8% agarose in 1x TBE (90 mM Tris–borate, 2 mM EDTA, 2.5 h at 90 V), transferred to nitrocellulose Hybond N+ (Amersham) and exposed to X-ray film.

2.5 Protein isolation
For isolation of whole protein extracts, hearts were homogenized in liquid nitrogen and lysed in RIPA buffer (50 mM Tris, pH 8, 1% Nonidet P40, 150 mM NaCl, 0.1% SDS, 0.5% deoxycholic acid) for 30 min on ice. After centrifugation at 20 000 g for 15 min, the protein content was determined by Bradford assay (Bio-Rad, München, Germany) using BSA as standard.

2.6 Western blot
Protein homogenates (60 µg for bcl-2 and bax, 100 µg for p17) were separated by SDS–polyacrylamide gel electrophoresis (bcl-2/bax 12%, p17 15%) and were blotted on PVDF membranes (Millipore, Schwalbach/Taunus, Germany) by a semidry blotting system (3 mA/cm2 for 45 min in buffer consisting of 48 mM Tris–HCl, 39 mM glycine, 0.037% SDS and 20% methanol). Unspecific binding was blocked (Bcl-2/Bax: 5% non fatty dry milk powder in TBS–Tween (50 mM Tris–HCl, pH 8, 150 mM NaCl, 2.5 mM KCl and 0.1% Tween 20); p17: 3% BSA–3% FCS in TBS–Tween). After incubation with primary antibodies against bcl-2/bax (1:500 in no-fatty dry milk, 2 h) and p17 (1:75 in 3%BSA–3% FCS, 1 h) blots were washed three times in TBS–Tween and than incubated with horseradish peroxidase-conjugated secondary antibody for 1 h (bcl-2/bax anti-rabbit, 1:4000; p17 anti-goat, 1:6000). Enhanced chemiluminesence was performed according to the instructions of the manufacturer.

2.7 Cloning of caspase-3 and detection of caspase-3 cleavage activity
Human caspase-3 was amplified by polymerase chain reaction (PCR) with oligonucleotides that were synthesized to contain BamH1 and SacI restriction sites and was cloned into the respective sites of pcDNA3.1 (Invitrogen, Leek, The Netherlands). Verified clones were in vitro transcribed/translated using the T7-polymerase kit (Promega) in the presence of 35S-labeled methionine. Then, degradation was determined by incubation of 0.5 µl [35S]-labelled caspase-3 with 150 µg of rat hearts homogenates (37°C, 24 h). As positive control 80 µg of Jurkat cell extracts, preincubated for 3 h with 2 µg/ml anti-Fas (Immunotech, Hamburg, Germany), were used. The resulting cleavage products were separated on 15% SDS–polyacrylamide gels and dried gels were exposed to X-ray films.

2.8 Statistics
Data are expressed as mean+S.D. Statistical analysis were performed with ANOVA followed by a modified least significant difference (Bonferroni) test using SPSS software.


   3 Results
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
4 Discussion
 Acknowledgments
 References
 
3.1 Apoptosis in ischemia and reperfusion
Apoptosis was determined by immunohistochemical detection of DNA strand breaks by TUNEL-staining of paraffin-embedded sections. Ischemic hearts did not reveal an enhanced appearance of TUNEL-positive cells (0.13±0.2%) compared to control hearts (0.12±0.11%). Reperfusion induced a 8.3-fold increase in TUNEL-positive cardiomyocytes (1±1.7%) as illustrated in Fig. 1. These results were confirmed by demonstration of the DNA laddering, a hallmark of apoptotic cell death. Thus, reperfusion induced DNA-fragmentation, whereas ischemia had no effect (Fig. 2)


Figure 1
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  		Fig. 1 Effect of inhibition of NOS on apoptosis during ischemia–reperfusion: TUNEL staining of isolated perfused rat hearts after ischemia (30 min) with or without reperfusion (30 min) in the presence or absence of 1 mM L-NMMA (see Methods). All photographs were taken at the same magnification (40x). Representative results are shown (n=6 per group).

 

Figure 2
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  		Fig. 2 DNA laddering: DNA was isolated from perfused rat hearts after ischemia (30 min) followed by reperfusion (30 min) in the presence or absence of 1 mM L-NMMA. After radioactive labeling the DNA was loaded onto agarose gels. Ethidium bromide staining confirms equal loading (upper panel) and DNA laddering was visualised by autoradiography (lower panel). A representative autoradiography is shown (n=6/group).

 
3.2 Inhibition of NOS augments apoptosis in ischemia–reperfusion
To eludicate the role of endogenous NO on apoptosis in ischemia–reperfusion, the NOS was inhibited by the competitive antagonist L-NMMA. As shown in Fig. 1, application of L-NMMA resulted in a drastic increase in TUNEL-positive cells, when hearts were exposed to ischemia (31±7% TUNEL-positive cells) and reperfusion (26±19% TUNEL-positive cells). Again, these immunohistochemical data were confirmed by detection of the DNA fragmentation. Thus, marked DNA-laddering was detected in ischemia–reperfusion, when the synthesis of endogenous NO was inhibited by L-NMMA (Fig. 2). L-NMMA alone elicited a minor pro-apoptotic effect (Fig. 2).

3.3 Effects of NOS inhibition on apoptosis signal transduction
The expression of Bcl-2 and Bax protein has been suggested to play an important pathophysiological role in the induction of myocyte apoptosis after ischemia and/or reperfusion [30]. Having demonstrated that inhibition of endogenous NO significantly enhanced ischemia- and reperfusion-induced apoptosis, we determined the effect of L-NMMA on Bcl-2 and Bax protein levels.

Western blots revealed a slight, but not significant increase of Bcl-2 protein levels in ischemia and reperfusion (Fig. 3A/B). Further inhibition of the NOS did not affect the Bcl-2-protein levels in ischemia or reperfusion (Fig. 3A/B).


Figure 3
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  		Fig. 3 Bcl-2 and Bax protein expression: (A) protein was obtained from rat hearts after ischemia (30 min) and following reperfusion (30 min) in the presence or absence of 1 mM L-NMMA. BCl-2 protein levels were assessed by Western blot with Bcl-2 antibodies. The blots were then reprobed with anti-Bax antibodies. Representative blots are shown from n=6 animals/group. (B) Protein expression was quantified densitometrically. Data are mean±S.D. with n=6 per group.

 
The pro-apoptotic protein Bax has been shown to be upregulated in neuronal ischemia [31,32]. However, in ischemic and reperfused hearts, no significant increase was detected either in the presence or the absence of L-NMMA, as shown in Fig. 3A/C.

Furthermore, we determined the activation of the caspase cascade by Western blot analysis of the active p17 subunit of caspase-3, which indicates the proteolytic activation of caspase-3. As illustrated in Fig. 4A, a minor increase of p17 was detectable after reperfusion. Furthermore, inhibition of the NOS in combination with ischemia–reperfusion synergistically enhanced the appearance of the active subunit.


Figure 4
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  		Fig. 4 Caspase-3 cleavage: protein was obtained from rat hearts after ischemia (30 min) and following reperfusion (30 min) in the presence or absence of 1 mM L-NMMA. (A) Western blot with antibodies raised against p17 subunit of caspase-3; a representative blot is shown from n=6 animals/group. (B) Cleavage of [35S]-labelled caspase-3 by protein homogenates of jurkat cells (pretreated with anti-Fas antibody 1 µg/ml for 6 h) or isolated reperfused hearts (ischemia–reperfusion in the presence of L-NMMA). After incubation of [35S]-labelled caspase-3 with protein homogenates at 4°C (control) or 37°C to allow for protease activity, proteins were separated by SDS-gel electrophoresis. A representative autoradiography from three independent experiments is shown.

 
To directly determine the caspase-3 cleavage activity, an in vitro assay was developed using exogenous 35S-labelled caspase-3 as substrate. As shown in Fig. 4B, protein extracts from anti-Fas activated Jurkat cells, the classical apoptosis in vitro system, were used as a positive control to induce the cleavage of 35S-labelled caspase-3. Moreover, protein extracts from ischemic–reperfused hearts, which were treated with L-NMMA revealed an enhanced caspase-3 activity, as demonstrated by the appearance of the radioactively labelled p17 cleavage fragment (Fig. 4B).


   4 Discussion
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
Acknowledgments
 References
 
The results of the present study demonstrate that inhibition of endogenous NO synthesis increased apoptosis during ischemia. Inhibition of NO synthesis thereby correlated with enhanced caspase-cleavage activity, whereas Bcl-2 and Bax protein levels were essentially unchanged. Thus, endogenous NO seems to suppress apoptosis of cardiomyocytes by interfering with the caspase cascade.

In the absence of the NOS inhibitor, 30 min of ischemia did not induce significant apoptotic cell death, whereas additional reperfusion resulted in a >8-fold increase of TUNEL-positive cells. Moreover, in reperfused hearts, the typical DNA-laddering, a hallmark of apoptotic cell, death was detectable. The increased apoptosis was due to apoptotic cell death of cardiomyocytes rather than inflammatory cells such as monocytes or lymphocytes, since the experiments were performed in isolated perfused hearts. Thus, this experimental approach allows to evaluate the direct effect of ischemia–reperfusion on cardiomyocytes in the absence of additional effects induced by invading inflammatory cells. Overall, the demonstration that reperfusion induces apoptosis, whereas ischemia alone reveals minor effects are in line with previous publications [5–7]. Controversially, one study demonstrated that ischemia without additional reperfusion resulted in increased apoptosis with maximal levels after 30 min of ischemia [33].

Reperfusion-induced cardiomyocyte apoptosis did not correlate with modulation of the protein levels of the Bcl-2-like proteins, Bcl-2 and Bax. In contrast to the Bcl-2 down-regulation observed in cerebral ischemia [31], the results of the present study demonstrate that Bcl-2 is not reduced after 30 min ischemia and 30 min reperfusion in isolated rat hearts. Moreover, Bax protein levels also remained constant. These findings are in contrast to previous studies, which demonstrated elevated Bax expression in myocardial infarction [30] and cerebral ischemia [31,32,34]. The increased Bax expression demonstrated in these studies may be due to invading activated inflammatory cells, which express high levels of Bax protein [28]. Since our studies were performed in isolated hearts in the absence of invading inflammatory cells, we directly assessed the effect of ischemia–reperfusion on cardiomyocytes. Thus, our present data suggest that Bax/Bcl-2 levels are not directly involved in cardiomyocyte apoptosis induced by ischemia–reperfusion injury. However, we demonstrated that caspases are activated by reperfusion. The active p17-subunit of the caspase-3 was detected after 30 min reperfusion, indicating proteolytical activation of caspase-3. This is in line with a recent report, which demonstrates the causal involvement of caspases in ischemia–reperfusion-induced apoptosis [35]. Moreover, caspase inhibitors were shown to reduce the infarct size suggesting an important role of caspase-dependent apoptosis in myocardial injury [35].

Finally, the results of the present study demonstrate that inhibition of endogenous NO synthesis enhances ischemia-induced cell death and this stayed unchanged during reperfusion. Thus, endogenous NO protects cardiomyocytes against apoptotic cell death. The anti-apoptotic effect of NO is supported by previous findings showing that L-arginine [23] and NO donors [24] are capable of preventing cardiomyocyte cell death induced by oxygen radicals. Other studies, however, suggest that the induction of iNOS exerts pro-apoptotic effects in cardiac myocytes [18,36]. Furthermore, inhibition of endogenous NOS prevented apoptosis [37] and led to a faster recovery of mechanical cardiac function in reperfusion [38]. Moreover, in cardiac allograft rejection, cardiomyocyte apoptosis was induced by NO [39]. These differences might be explained by the different experimental settings used. In our experiments, iNOS induction is unlikely to occur since the ischemic period of 30 min is to short to induce iNOS expression. Indeed, iNOS could not be detected by Western blot (data not shown) and, therefore, cannot contribute to the rapid induction of cardiomyocyte apoptosis in ischemia–reperfusion.

The induction of apoptosis by inhibition of NO synthesis in ischemia appears to be due to the activation of the caspase cascade. Thus, caspase-3 cleavage activity was enhanced in L-NMMA-treated hearts. Moreover, the active p17 subunit of caspase-3 was detected by Western blot. This might be explained by NO-mediated basal inhibition of caspase-activity due to S-nitrosation of the essential cysteine residue, which has been demonstrated in vitro [21] and in vivo [25]. Removal of NO may enhance basal caspase-3-activity and thus sensitise the cells towards pro-apoptotic insults. This is in line with a slight increase of DNA fragmentation in control hearts induced by L-NMMA-treatment. In conclusion, NO released by the constitutive NOS seems to be protective against cardiomyocyte apoptosis by interference with the activation of the caspase cascade.

Time for primary review 23 days.


   Acknowledgments
Top
 Abstract
 1 Introduction
 2 Methods
 3 Results
 4 Discussion
 Acknowledgments
References
 
This study was supported by grants from the Deutsche Forschungsgemeinschaft SFB 553 (Teilprojekt C2).


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

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