© 1999 by European Society of Cardiology
Copyright © 1999, European Society of Cardiology
Induction of necrosis but not apoptosis after anoxia and reoxygenation in isolated adult cardiomyocytes of rat
Physiologisches Institut, Justus-Liebig-Universität Giessen, Giessen, Germany
* Corresponding author. Tel.: +49-641-994-7243; fax: +49-641-994-7239; e-mail: gerhild.taimor@physiologie.med.uni-giessen.de
Received 14 November 1997; accepted 3 June 1998
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Abstract |
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Objectives: Apoptosis is one feature of myocardial damage after ischemia–reperfusion, but the causes for its induction are unclear. The present study was undertaken to investigate whether apoptosis in cardiomyocytes is directly initiated by their sub-lethal injury that results from ischemia–reperfusion. Methods: Ischemia was simulated on isolated ventricular cardiomyocytes of adult rats by anoxia in a glucose free medium, pH 6.4. Induction of apoptosis was detected by (1) DNA laddering of genomic DNA, (2) TUNEL positive cells (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling) and (3) annexinV–fluorescein isothiocyanate (annexinV–FITC) binding to cells under exclusion of propidium iodide. Necrotic cells were identified by (1) staining with both annexinV–FITC and propidium iodide, (2) unspecific DNA degradation and (3) enzyme release. Results: Simulated ischemia caused a >75% loss of high-energy phosphates within 2 h, which was reversible upon reoxygenation at pH 7.4. Even after 18 h of simulated ischemia, creatine phosphate contents recovered to 55.2±7.3% of control within 1 h. Apoptosis could be induced by UV irradiation (80 J/m2), H2O2 and the NO-donor N2-acetyl-S-nitroso-D,L-penicillinaminamide. In contrast to this, simulated ischemia and reoxygenation could not induce apoptosis in the cells, but with prolonged ischemia more cells became necrotic. After 18 hours of simulated ischemia and 4 h of reoxygenation 41.2±10.2% myocytes were necrotic (vs. 6.3±4.4% of control) and only 1.7±0.5% (vs. 8.7±4.6% of control) were apoptotic. The percentage of necrotic cells correlated with an increase in lactate dehydrogenase release from 9.9±0.6% (of total activity) of normoxic controls to 37.9±5.1% after 18 h of simulated ischemia and 12 h of reoxygenation. Conclusions: Simulated ischemia-reoxygenation causes necrosis of isolated cardiomyocytes but is not sufficient for induction of apoptosis.
KEYWORDS Apoptosis; Anoxia; Cell culture; Energy-metabolism; Necrosis
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 DiscussionReferences |
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Ischemia, caused by the reduction of coronary blood flow results in death of cardiac tissue. This can occur in the form of necrosis or apoptosis. Necrotic cell death is a consequence of severe cell damage and is not transcriptionally controlled. It is characterised by loss of plasma membrane integrity, followed by enzyme release and unspecific DNA degradation [1]. In contrast, apoptosis is a transcriptionally controlled cellular response to moderate cell damage or various cytokines [2–4]. In cells undergoing apoptosis the chemical structure of the plasma membrane changes, but its physical integrity is initially preserved [5]. Due to activation of endonucleases genomic DNA is cut internucleosomally, resulting in DNA oligomeres of about 180 base pairs and multiples thereof (‘DNA laddering') [1].
Investigations of autopsy cases after myocardial infarction [6, 7]and of in situ ligated hearts [8, 9]demonstrate the appearance of apoptosis besides necrosis in ischemic and reperfused myocardium. These observations have raised questions about the nature of inducers and the mechanisms leading to apoptosis in ischemic or ischemic-reperfused myocardium. Apoptosis might be directly initiated by the sub-lethal injury of cardiomyocytes in an energy depleted state or, alternatively, by exogenous apoptosis-promoting factors released from non-myocytes in ischemic–reperfused tissue. The present study was undertaken to investigate the first hypothesis, i.e., whether induction of apoptosis has an endogenous cause. In whole myocardial tissue it is impossible to separate causes that are endogenous to the myocardial cell from those imposed on the myocardial cell from outside. Therefore, we used isolated cardiomyocytes submitted to simulated ischemia–reperfusion as an experimental model, which has been characterised by our group in detail before. It has been shown that, in this model, cardiomyocytes are energy depleted within the first hour of ischemic conditions [10]. Similar to the in vivo situation, energy depleted cardiomyocytes develop rigor contracture and cytosolic Ca2+-overload [11]. Reoxygenation results in a rapid recovery of the cellular state of energy and Ca2+-control, but also in hypercontracture of the cells [10]. The question whether simulated ischemia and reoxygenation would be able to directly induce apoptosis in cardiomyocytes was investigated in this model.
In control experiments, testing the ability of cardiomyocytes to undergo apoptosis, the cells were exposed to UV irradiation. Apoptotic cell damage was monitored by three indicators: (i) the appearance of DNA laddering in agarose gels, (ii) the TUNEL assay, detecting the specific DNA breakdown in situ, and (iii) the translocation of phosphatidylserine to the outer cell surface, detected by binding of FITC-labelled annexinV under exclusion of the DNA dye propidium iodide [5, 12–14]. As indications of necrotic cell death (i) cellular enzyme release, (ii) unspecific DNA degradation and (iii) simultaneous staining of the cells with annexin–FITC and propidium iodide were determined. Simulated ischemic conditions were established by incubation of cells in anoxic, glucose-free medium with acidotic pH 6.4. After various times of simulated ischemia cells were reoxygenated in medium with pH 7.4 (simulated reperfusion) to allow the full expression of either apoptosis or necrosis.
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2 Methods |
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The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1985.)
2.1 Cell isolation and short-term cultures
Ventricular cardiomyocytes were isolated from 200–250 g male Wistar rats, suspended in basal culture medium and plated on 60 mm culture dishes, which were preincubated over night with 4% fetal calf serum in medium 199 as previously described [15]. The basal culture medium (CCT) was modified medium 199 including Earle's salts, 2 mM L-carnitine, 5 mM taurine, 100 IU/ml penicillin, 100 µg/ml streptomycin and 10 µM cytosine-β-D-arabinofuranoside (pH 7.4). Three hours after plating the dishes were washed twice with modified phosphate free Tyrode's medium (140 mM NaCl; 3.6 mM KCl; 1.2 mM Mg2SO4; 1 mM CaCl2 and 20 mM N-2-(hydroxyethyl)-piperazine-N'-2-ethansulfonic acid, pH 7.4). As a result of the medium change broken cells were removed resulting in cultures with 93±2% quiescent rod-shaped cells.
2.2 Experimental protocols
For simulated ischemia dishes were filled with 1 ml of the modified Tyrode's medium (pH 6.4), gassed with 100% N2 and incubated at 37°C in gas tight chambers in an atmosphere of 100% N2. Reoxygenation was performed by addition of 1 ml CCT medium and incubation of the cells at 37°C with air oxygen. Time matched controls for anoxic incubations were obtained by use of air equilibrated instead of N2 saturated media with pH 7.4.
In one set of experiments cells were energy depleted by chemical blockade of oxidative phosphorylation as described by Gottlieb et al. [16]. Cells were incubated for 30 minutes in a KCN containing acidotic buffer (106 mM NaCl, 4.4 mM KCl, 1.0 mM MgCl2, 38 mM NaHC03, 2.5 mM CaCl2, 20 mM 2-deoxyglucose, 1.0 mM KCN; pH 6.6) at 37°C. For metabolic recovery this buffer was substituted by CCT medium.
As a control stimulus for induction of apoptosis cardiomyocytes were irradiated with 254 nm UV light (UVC) at 80 J/m2. For attenuation of apoptotic cell death cardiomyocytes were preincubated for 1 h with the caspase-3 inhibitor Z-DEVD-FMK (10 µM) and UV irradiation and reculturing were also done in the presence of the inhibitor.
2.3 Determination of creatine phosphate and ATP contents
Experiments were terminated by addition of 1 ml 1.2 M HClO4 to the cultures. Protein was determined in the acid precipitates according to Bradford [17]using bovine serum albumin as standard. After neutralisation, perchloric acid extracts of cultures were analysed for ATP and creatine phosphate (CrP) [18].
2.4 Analysis of genomic DNA
12 h after UV irradiation or simulated ischemia DNA was extracted as described by Tanaka et al. [19]. Cardiomyocytes were harvested by centrifugation at 2800 g for 5 minutes. After resuspension in lysis buffer (100 mM NaCl; 10 mM Tris/Cl; 25 mM ethylenediamine tetraacedic acid (EDTA); 0.5% sodium dodecyl sulfate, 100 µg/ml proteinase K, pH 8.0) myocytes were incubated for 3 h at 37°C. After phenol/chloroform extraction and ethanol precipitation DNA was dissolved in TE buffer (10 mM Tris/HCl, 1 mM EDTA, pH 8.0) and incubated with 5 µg/ml DNAse-free RNAse for 2 h at 37°C. Again the DNA was precipitated and resuspended in TE buffer. Concentrations were measured spectophotometrically. 4 µg of each DNA were electrophoretically separated on 1.5% agarose gels and stained with ethidium bromide.
2.5 TUNEL assay (terminal deoxynucleotidyl transferase-mediated-UTP nick end labelling)
Cardiomyocytes were fixed in 70% ethanol at –20°C for at least 2 h, followed by fixation in 4% paraformaldehyd for 10 min at room temperature. After rinsing with PBS (4 mM NaH2PO4, 16 mM Na2HPO4, 150 mM NaCl; pH 7.4) the labelling reaction was performed 60 min at 37°C in the presence of 0.1 U/µl terminal deoxynucleotidyl transferase and 20 pmol/µl biotin-16-dUTP. The cells were washed with PBS and incubated for 30 min in 0.6 M NaCl, 60 mM Na-citrate, 5% nonfat dry milk, pH 7.0. Apoptotic nuclei were then stained with streptavidin–Texas Red for 30 min. For quantification of apoptosis 100 randomly distributed cells were counted in each experiment.
2.6 AnnexinV-binding assay
AnnexinV–FITC/propidium iodide staining was performed essentially as described by Vermes et al. [13]and Martin et al. [14]. In detail, 10 µl of annexinV–FITC (Boehringer Ingelheim) and 1 ng propidium iodide were added to the culture medium (2 ml) between 2 h and 4 h after damage. After incubation for 10 min at 37°C in the dark cells were washed with PBS and analysed by fluorescence microscopy. An early effect of apoptosis is the translocation of phosphatidylserine from the inner site of the plasma membrane to the outer surface [5]while the membrane remains physically intact. Apoptotic cells therefore can be stained with annexinV–FITC, which binds with high affinity to phosphatidylserine, resulting in a green fluorescence when excited at 450–480 nm. At the same time, propidium iodide, a DNA dye incapable of passing the plasma membrane is excluded (ANX+/PI–). Necrotic cells have lost the physical integrity of their plasma membrane and are therefore stained with both annexinV–FITC and propidium iodide which fluoresces in the red when exited at 510–550 nm (ANX+/PI+). Cells which are neither apoptotic nor necrotic do not stain with either dye. For quantification of apoptosis and necrosis 100 randomly distributed cells were counted in each experiment. The small number of necrotic cells at time zero was subtracted from all following counts.
2.7 Determination of lactate dehydrogenase release
The activity of lactate dehydrogenase (LDH) in the culture medium and in cardiomyocytes, which were lysed by addition of TritonX-100 (1% final concentration) was determined 12 h after intervention according to Bergmeyer and Bernt [20]. Total activity was defined as the sum of enzyme activity in media and in TritonX-100 extracts. The validity of this approach was tested in experiments in which we found that total activity did not change in 30 h cultures with intact or TritonX-100 lysed cells. The release of enzymes in the medium was calculated as the percentage of enzyme activity in the media compared to total activity of the cultures. Enzyme release during the first hour, related to the small number of initially necrotic cells, was subtracted from all following values.
2.8 Statistics
Data are given as means±standard deviations (SD) from n different culture preparations. In each culture 100 randomly distributed cardiomyocytes were counted to obtain the percentage of apoptotic (ANX+/PI–) or necrotic cells (ANX+/PI+) after annexinV–FITC/propidium iodide staining. Statistical comparisons were performed by one-way analysis of variance (ANOVA) and use of Student Newman Keuls test for post hoc analysis [21, 22]. A p-value less than 0.05 was considered to indicate statistical significance. All data analyses were computed using SAS® software, version 6.11 (SAS Institute Inc., Cary, N.C., USA).
2.9 Materials
Medium 199 and terminal deoxynucleotidyl transferase was obtained from Boehringer (Mannheim, Germany), fetal calf serum from PAA (Linz, Austria), crude collagenase was from Biochrom (Berlin, Germany), Z-DEVD-FMK from Calbiochem (Heidelberg), streptavidin- texas-red was from Amersham (Braunschweig, Germany) and annexinV–FITC was from Boehringer (Ingelheim, Germany).
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3 Results |
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3.1 High energy-phosphates and enzyme release during simulated ischemia and reoxygenation
Under normoxic conditions cardiomyocytes contained 27.2±2.4 nmol CrP/mg protein and 24.4±2.1 nmol ATP/mg protein (Fig. 1). During conditions of simulated ischemia cardiomyocytes progressively became depleted of high-energy phosphates. After 2 h they reached levels of 5.2±2.1 nmol CrP/mg protein and 5.0±2.0 nmol ATP/mg protein. When the cells were reoxygenated after 2 h of simulated ischemia they completely recovered their CrP to 24.0±2.4 nmol/mg protein during 1 h of reoxygenation (Fig. 1). Reoxygenation for 1 h after 18 h of simulated ischemia brought CrP contents back to 15.0±2.0 nmol/mg protein. Once recovered, CrP concentrations remained constant during 12 h of reoxygenation. Recovery of ATP contents was small even after the first 2 h of simulated ischemia.
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As a sign of irreversible damage of cardiomyocytes caused by the loss of plasma membrane integrity the activity of LDH released from cells was measured and related to the total enzyme activity per culture dish, which remained constant during 30 h of cell culture. Control cells, which were incubated under normoxic conditions for the same time span (30 h-control) as the longest simulated ischemia and reoxygenation lasted, released 9.9±0.6% of total LDH activity into the medium (Fig. 2). The released LDH activity increased continuously with time of simulated ischemia in reoxygenated cultures. After 18 h of simulated ischemia and 12 h of reoxygenation the release of LDH activity amounted to 37.9±5.1% of total LDH activity. In contrast, enzyme release of cardiomyocytes 12 h after UV irradiation (12.3±1.9% of total activity) showed only a small increase as compared to the 12 h-control (7.2±1.6% of total activity). Similar results were obtained for release of creatine kinase (data not shown).
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3.2 Apoptosis of UV irradiated cardiomyocytes
As a control stimulus for induction of apoptosis cardiomyocytes were damaged by UV irradiation, a well known inducer of apoptosis. For this purpose cardiomyocytes were irradiated with 254 nm UV light (UVC) at 80 J/m2.
For quantification of apoptosis and necrosis cells were stained with annexinV–FITC/propidium iodide different times after UV irradiation. The percentage of cardiomyocytes exhibiting the apoptotic pattern of labelling with annexinV–FITC (ANX+/PI–) was already increased 1 h after UV irradiation, and 2 h to 4 h after damage the number of apoptotic cardiomyocytes reached a constant level of 16.9±4.5% 4 h after irradiation as compared to 4.7±3.0% in time matched controls (Fig. 3). The number of double-stained necrotic cells (ANX+/PI+) did not rise significantly. Therefore quantification of cell death by the annexinV-binding assay was always performed 4 h after damage. A typical fluorescence microscopic picture of cardiomyocytes after UV irradiation is shown in Fig. 4. In contrast to annexinV–FITC binding, induction of DNA laddering was delayed. It became evident on agarose gels 6 h after UV irradiation but was stronger after 12 h (Fig. 5, lanes 4 and 5). Therefore DNA laddering was always monitored 12 h after damage. Because the TUNEL assay also detects the specific DNA breakdown in the cells this assay was applied after the same time. Using this method 12.6±4.9% apoptotic cells were found 12 h after UV irradiation as compared to 5.4±0.7% in time matched controls. The two labelling techniques allowing quantification of cell death thus gave similar results.
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-DNA, HindIII digested. DNA was stained by ethidium bromide.The influence of simulated ischemia on the induction of apoptosis by UV irradiation was also studied. When cardiomyocytes were first UV irradiated and then exposed to 2 h or 4 h of simulated ischemia and 4 h of reoxygenation, the number of apoptotic cells (ANX+/PI–) raised to the same extent (18.1±3.6% after 4 h of simulated ischemia/reoxygenation) as upon UV irradiation followed by 4 h of continuous normoxic conditions (16.9±4.4%) (Fig. 3). The number of necrotic cells (13.8±7.3%) was not influenced by UV irradiation as compared to the respective control (14.3±5.5% after 4 h of simulated ischemia/reoxygenation). This demonstrates that a period of up to 4 h of simulated ischemia does not suppress induction of apoptosis nor turn an apoptotic pattern of cell injury into a necrotic one. In all ischemia experiments, therefore, development of apoptosis and necrosis was monitored in 4 h time intervals.
In the presence of the caspase-3 inhibitor Z-DEVD-FMK the induction of apoptosis by UV irradiation was attenuated from 16.9±4.5% to 9.2±3.3% apoptotic cells (ANX+/PI–) (Fig. 6). The inhibitor had no effect on the low percentage of necrotic cells (ANX+/PI+) in the preparation. In further experiments (see below) it was therefore used to differentiate between primary necrosis and necrosis resulting secondarily from apoptosis.
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It was also tested if apoptosis can still be induced by UV irradiation after prolonged energy depletion. For that purpose, after 18 h of simulated ischemia and 1 h of reoxygenation cardiomyocytes were UV irradiated. 4 h after UV irradiation cells were stained. The percentage of apoptotic cells (ANX+/PI–) significantly increased to 10.1±1.9% as compared to 2.1±0.6% in time matched, non-irradiated controls (Fig. 6). This experiment shows that even after 18 h of simulated ischemia an apoptotic response can be evoked in cardiomyocytes.
3.3 Necrosis and apoptosis during simulated ischemia and reoxygenation
Cardiomyocytes were exposed to simulated ischemia between 2 h and 18 h followed by 12 h of reoxygenation before DNA extraction. Fig. 7A demonstrates that DNA laddering was detectable only in the UV irradiated controls (lane 3), which were harvested 12 h after UV damage. During the first 6 h of simulated ischemia followed by reoxygenation the DNA of myocytes remained intact (lanes 4 and 5). After longer times of simulated ischemia and reoxygenation the DNA starts smearing over the gel (lanes 6–8), which is a sign of unspecific DNA degradation and necrotic cell death. 18 h of simulated ischemia without reoxygenation resulted in the same extent of unspecific DNA degradation (Fig. 7B).
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For quantification of apoptosis and necrosis by annexinV–FITC/propidium iodide staining, cardiomyocytes were submitted to simulated ischemia for up to 18 h, followed by 4 h of reoxygenation before staining. Time intervals of 1 to 4 h during simulated ischemia were chosen to make sure that an early induction of apoptosis did not escape detection. In the normoxic controls we found 8.7±4.6% apoptotic (ANX+/PI–) and 6.3±4.4% necrotic cardiomyocytes (ANX+/PI+) (Fig. 8). Up to 3 h of simulated ischemia and following reoxygenation no changes in the number of apoptotic or necrotic cells were seen. After four and more hours of simulated ischemia and subsequent reoxygenation the number of necrotic cardiomyocytes (ANX+/PI+) increased continuously, reaching 41.2±10.2% after 18 h of anoxia and 4 h of reoxygenation. The number of apoptotic cells did not increase, but in fact decreased. An increase in apoptotic cells after simulated ischemia and reoxygenation was not found in the TUNEL assay either (data not shown).
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Although we already demonstrated that the time intervals chosen under ischemic conditions are sufficiently small for detection of induced apoptosis, it was independently investigated whether the observed rise in necrotic cell death could, at least in part, have developed secondarily from apoptotic cells. Experiments with simulated ischemia and reoxygenation were performed in the presence of the apoptosis inhibitor Z-DEVD-FMK (Fig. 9). In its presence the number of apoptotic and necrotic cells was not found to be changed.
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3.4 Inducers of apoptosis in cardiomyocytes
Cardiomyocytes were energy depleted by metabolic inhibition of oxidative phosphorylation with 1 mM KCN for 30 min. Anaerobic substrate chain phosphorylation was blocked by addition of 20 mM 2-deoxyglucose. The inhibitors were then removed by a complete change of the medium. After another 4 h incubation without inhibitors apoptotic and necrotic cells were quantified. We found 25.5±7.8% apoptotic cardiomyocytes (ANX+/PI–) as compared to 4.7±1.3% under control conditions (Table 1). The low number of necrotic cells remained unchanged.
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It was also tested whether cardiomyocytes can be driven into apoptosis by chemical radical generators. Addition of the NO-donor SNAP (N2-acetyl-S-nitroso-D,L-penicillinaminamide) or the oxidant H2O2 resulted in an increase in the number of apoptotic cells. Incubating the cardiomyocytes for 2 h with 100 µM SNAP induced 14.1±1.9% apoptotic cells (ANX+/PI–), whereas the inactive SNAP-analog NAP (nitroso-D,L-penicillinaminamide) did not damage the cells (4.8±0.3% ANX+/PI–). 10 µM H2O2 increased the level of apoptosis to 14.7±5.1% after 2 h (Table 1). Both agents, SNAP and H2O2, induced DNA laddering (data not shown).
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4 Discussion |
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The central question of the present study has been whether cardiomyocytes exposed to simulated ischemia-reoxygenation would spontaneously develop apoptosis. The main finding is that neither the metabolic depression under conditions of simulated ischemia nor the process of metabolic alterations in reoxygenation are sufficient endogenous causes for induction of apoptosis in isolated adult cardiomyocytes. Experiments using UV irradiation, a NO donor, oxidative stress or metabolic inhibition under normoxic conditions demonstrated that cardiomyocytes are capable of an apoptotic reaction.
The model of isolated ventricular cardiomyocytes from rat exposed to simulated ischemia and subsequent reoxygenation has been characterised in detail in previous studies [10, 11, 23]. As now confirmed, cardiomyocytes are depleted of high energy phosphates within 2 h of oxygen- deprivation. Oxygen-deprived cardiomyocytes retain their metabolic competence for oxidative phosphorylation for a long period of time, as demonstrated by the recovery of CrP levels upon reoxygenation. This recovery of CrP depends on the restoration of a normal free energy change of ATP hydrolysis, i.e. on activation of oxidative energy production in mitochondria [24]. The absolute contents of ATP remain low since anoxic-reoxygenated cells lose most of their purine contents after simulated ischemia and reoxygenation and possess only a very small capacity for their de novo synthesis.
Besides the often used parameters of enzyme release for detection of necrosis and DNA laddering for apoptosis, we used annexinV–FITC/propidium iodide staining of the cells for differentiation and quantification of the two processes of cell deterioration. Phosphatidylserine externalisation, which can be detected by this assay, has been shown to be an early event during apoptosis in a variety of murine and human cell types, regardless of the stimulus inducing apoptosis [14]. And the assay gives similar results as other methods for apoptosis detection [13]. In a set of basic experiments of the present study a close correspondence between the increase in the number of ANX+/PI- cardiomyocytes and the detection of TUNEL-positive cells as well as the appearance of DNA laddering was shown.
In normoxic controls we found approximately 9% apoptotic and 6% necrotic cardiomyocytes. These values are likely due to the stress of cell isolation. Increasing times of simulated ischemia followed by reoxygenation produced an increasing number of necrotic cells. This became apparent by unspecific DNA degradation and by the increasing number of cells stained with both annexinV–FITC and propidium iodide. After 18 h of simulated ischemia and 4 h of reoxygenation, approximately 40% of the individual cells showed these histological markers of necrosis. This corresponded to a 38% loss of the cytosolic enzyme lactate dehydrogenase from the cell population and a decrease in metabolic competence of the cultures after 18 h of simulated ischemia, which recovered only 55% of creatine phosphate reserves upon reoxygenation. The main cause of necrotic cell death seems to be simulated ischemia, since DNA degradation after simulated ischemia was found independent of reoxygenation. It seems highly unlikely that a part of the necrotic cell death is secondary to a transient state of apoptosis, since, first, the temporal pattern of analysis was shown not to miss a once induced apoptosis and, second, presence of Z-DEVD-FMK, an effective apoptosis inhibitor, did not alter the development of necrosis during simulated ischemia/reoxygenation.
Several studies have revealed an induction of apoptotic cell death in ischemic-reperfused myocardium [6, 8, 9]. Comparison of our results, which indicate that simulated conditions of ischemia and reperfusion applied directly to the cardiomyocytes are not sufficient to induce apoptosis in these cells, with those obtained in complex cell and tissue systems leads to the suggestion that in whole heart muscle additional signals from non-myocytic cells participate in the induction of apoptosis. Influence of non-myocytic cells on the development of apoptosis in cardiomyocytes was indicated, e.g., by the study of Suzuki et al. [25]who showed a relationship between NO production in macrophages and induction of apoptosis in myocardium after coronary occlusion. In the present study we confirmed that a high dose of an NO-donor can induce apoptosis in isolated adult cardiomyocytes. It may be considered that in the isolated cardiomyocyte model loss of energy phosphates proceeds less rapidly than in the heart in vivo when exposed to conditions of ischemia. This delay is due to the lower energy demand of the isolated cardiomyocytes which are mechanically quiescent. However, it has been shown in several studies before that decisive features of ischemic intracellular changes, such as developement of energy depletion, acidosis and Ca2+ overload, and of reoxygenation injury, such as hypercontracture develop in the isolated cells [10, 11, 23]as well as in the intact myocardium.
Although simulated conditions of ischemia-reoxygenation did not induce apoptosis in isolated cardiomyocytes metabolic inhibition by cyanide treatment induced apoptosis as has also been shown by Gottlieb et al. [16]. One may speculate that in cyanide treated cardiomyocytes, which are left normoxic, oxidative stress is greater than in anoxic cells and that this stress causes apoptosis. In the experiments of Tanaka et al. [19], who found apoptosis induction in cultures of neonatal rat cardiomyocytes under hypoxic instead of anoxic conditions, this possibility can also not be excluded. Indeed, oxidative stress is a known inducer of apoptosis [3, 26]. In the present study, the oxidant H2O2 was found to induce apoptosis in cardiomyocytes. In heptatocyte cultures it has been shown that cell death caused by chemical inhibition is decreased when performed under hypoxic conditions [27]. And in ischemic hearts apoptosis is more profound in the border zone of infarction, where the oxygen concentration is higher [7, 28].
In conclusion, the present study has shown for the first time that neither simulated ischemia nor reoxygenation represent sufficient causes for directly inducing apoptosis in adult cardiomyocytes. The findings suggest that complex mechanisms are responsible for induction of apoptosis in the ischemic-reperfused myocardium.
Time for primary review 25 days.
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Acknowledgements |
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This study was supported by a grant of the BIOMED-2 program of the European Union.
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