© 2000 by European Society of Cardiology
Copyright © 2000, European Society of Cardiology
Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in the ischemia–reperfused rat heart
aSecond Department of Internal Medicine, Yamaguchi University School of Medicine, Yamaguchi, Japan
bSecond Department of Internal Medicine, Gifu, University School of Medicine, Gifu, Japan
* Corresponding author. Tel.: +81-836-22-2248; fax: +81-836-22-2246 masunori{at}po.cc.yamaguchi-u.ac.jp
Received 15 March 1999; accepted 20 August 1999
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Abstract |
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 Top Abstract 1 Introduction2 Methods3 Results4 Discussion5 ConclusionsAcknowledgmentsReferences |
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Objective: Caspase family proteases are recognized as key mediators of apoptosis. However, the role of caspases in the ischemia–reperfused heart remains uncertain. We evaluated the effect of caspase inhibitors on myocardial infarct size and the myocyte DNA fragmentation in the ischemia–reperfused rat hearts. Methods: Three groups of Sprague–Dawley rats (n=7, each) were subjected to 30 min of ischemia followed by 6 h of reperfusion. One of the following drugs: (1) YVAD-aldehyde, a caspase-1-like protease inhibitor (3.5 mg/kg; YVAD), (2) DEVD-aldehyde, a caspase-3-like protease inhibitor (3.5 mg/kg, DEVD), (3) vehicle (140 µl/kg) was administered intravenously 5 min prior to the ischemia in each group. Myocardial infarct size was defined by triphenyltetrazolium chloride (TTC) staining. Immunohistochemical staining by in situ nick end labeling (TUNEL) of cardiomyocytes and DNA electrophoresis were used for detecting DNA fragmentation. Ultrastructural analysis was done by electron microscopy. The caspase activity was measured in the myocardium of both groups. Results: The percentage of TUNEL-positive myocyte nuclei (%AP) was quantified by microscopy. A ladder pattern was detected by electrophoresis of DNA from the risk area and TUNEL-positive myocytes were seen in the risk area. The %AP was significantly reduced from 20±1% to 12±3% by YVAD and to 10±3% by DEVD (both P<0.01). However, caspase inhibitors did not significantly change the infarct size. Electronmicrograph showed similar salcolemmal and mitochondrial damage in both group. The caspase activity was blocked by DEVD at 4 h after reperfusion. Conclusion: Myocyte DNA fragmentation and caspase activation was inhibited by caspase inhibitors without reduction of the infarct size in ischemia–reperfused rat hearts.
KEYWORDS Apoptosis; Infarction; Myocytes
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1 Introduction |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionsAcknowledgmentsReferences |
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Ischemia–reperfusion insult induces myocardial infarction in the ischemic area. This infarcted area consists of necrotic cells as well as so-called apoptotic cells which shows characteristic DNA fragmentation [9]. However, the ultrastructural feature of the ‘apoptotic’ myocytes subjected to the ischemia–reperfusion insult demonstrated oncosis accompanied by DNA fragmentation [13].
A group of cysteine proteases, caspases, play key biological roles in inducing apoptosis. Caspase-1 (interleukin-1β converting enzyme; ICE) and caspase-3 (CPP32/Yama/apopain) are detected in cardiomyocytes. Caspase-1 recognizes a peptide sequence YVAD, cleaving after aspartate, whereas caspase-3 recognizes a peptide sequence DEVD and cleaves poly (ADP-ribose) polymerase (PARP) [12,17]. Caspases-1 and -3 constitute a protease cascade, where caspase-3 is a downstream effector protease leading to DNase activation [3,16] followed by DNA fragmentation [7,14].
In cardiomyocytes, caspase inhibitors can reduce incidence of apoptosis induced by metabolic inhibition in the isolated rabbit heart [10]. Recently, Yaoita et al. [21] showed attenuation of myocardial injury brought by a caspase inhibitor. However, the relationship between the infarct size and the incidence of DNA fragmentation is controversial. In this study, Ac-Tyr–Val–Ala–Asp-aldehyde (YVAD-CHO, an inhibitor of caspase-1-like proteases) or Ac-Asp–Glu–Val–Asp-aldehyde (DEVD-CHO, an inhibitor of caspase-3-like proteases) were administered before ischemia–reperfusion insult, and their effects on infarct size and the incidence of myocyte DNA fragmentation were evaluated. The caspase-3 activity was measured in the ischemic and non-ischemic myocardium and the effect of DEVD on the activity was evaluated.
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2 Methods |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionsAcknowledgmentsReferences |
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2.1 Animal preparation
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 Yamaguchi University School of Medicine. Male Sprague–Dawley rats (280 to 330 g) were anesthetized by intraperitoneal injection of sodium pentobarbital (60 mg/kg). Additional anesthesia was given during the experiment as necessary. After the tracheal intubation, the rat was ventilated by a respirator (Model SN-480-7, Astec, Japan) with a mixture of oxygen and room air (tidal volume, 1.2 ml/100 g; respiratory rate, 65–70/min). Arterial blood gases were measured and arterial PO2, PCO2 and pH were kept within a physiological range by adjusting the ventilator. After the thoracotomy in the fourth intercostal space, the heart was suspended on the pericardial cradle. A 6-0 silk thread was passed around the left anterior descending coronary artery near its origin using a tapered needle. Both ends of the thread were passed through a 1-cm long polyethylene tube (outer diameter, 1.5 mm) which was used to occlude the coronary artery by pulling the thread. Left ventricular pressure (LVP) was measured by a 2F catheter-tip manometer (Model SPC-320, Millar Instruments, USA) inserted via the right carotid artery. Peak and end-diastolic LVP and the first derivative of LVP (+dP/dt) were calculated by an on-line data acquisition system (CODAS, DATAQ Instruments, Ohio, USA). Myocardial ischemia was confirmed by regional cyanosis, bulging of the relevant segment of the left ventricle, an increase in the left ventricular end-diastolic pressure and ST elevation on ECG. Body temperature was measured by an electric thermistor placed in the rectum, and maintained at 37±0.3°C by a heating pad placed under the rat.
2.2 Reagents
YVAD-CHO and DEVD-CHO (Peptide Institute, Osaka, Japan) are competitive tetrapeptide inhibitors of caspase. YVAD-CHO inhibits caspase-1 and other subfamily members such as caspase-4, whereas DEVD-CHO inhibits caspase-3 and subfamily members such as caspase-7 and caspase-1 [4,18].
2.3 Experimental protocol
Rats were randomly assigned to three groups. A final volume of 300 µl of 15% dimethylsulfoxide (DMSO) diluted with saline was administered to the vehicle group (n=7), followed by a 30-min coronary occlusion and a 6-h reperfusion. YVAD-CHO (3.5 mg/kg) diluted in DMSO (300 µl) was administered intravenously to the YVAD group (n=7) 5 min before a 30-min coronary occlusion followed by a 6-h reperfusion. DEVD-CHO (3.5 mg/kg) diluted in DMSO (300 µl) was administered intravenously to the DEVD group (n=7) 5 min before a 30-min coronary occlusion followed by a 6-h reperfusion. After the 6-h reperfusion, rats were killed with an overdose of sodium pentobarbital.
2.4 Quantitation of infarct size
The left anterior descending coronary artery was reoccluded after the 6- h reperfusion and the heart was excised. Monastral blue dye (1.5%, 1 ml) was injected into the ascending aorta to delineate the risk area. The heart was cut parallel to the atrio-ventricular groove at the center of the risk area. The distal portion was immediately fixed with 10% formalin for subsequent in situ nick end labeling (TUNEL). The proximal portion was cut in 1-mm thick slices which were incubated in 1% triphenyltetrazolium chloride (TTC) at 37°C for 20 min. Viable myocardium was stained red and necrotic area remained unstained by TTC [19]. The slices were imaged by a color CCD camera (FV-10, Fuji, Japan). The images were stored on a computer and analyzed by NIH image. The area stained by a blue dye is identified as perfused area and the unstained area as an area at risk (AR). Infarcted area (AN) is identified as unstained area by TTC. AR was normalized by the whole left ventricular area as AR/LV and AN was normalized by AR as AN/AR.
2.5 In situ nick end labeling
The TUNEL protocol is based on the preferential labeling of terminal deoxynucleotidyl transferase at the 3'-OH ends of DNA [8]. In brief, the fixed transverse ventricular slices were embedded in paraffin and 4-µm thick sections were deparaffinized by washing in xylene and a descending ethanol series. The sections were subsequently incubated with 20 µg/ml proteinase K for 15 min at room temperature, and endogenous peroxidase was inactivated by a treatment of 3% hydrogen peroxide for 5 min. They were incubated with terminal deoxynucleotidyl transferase (ApopTag®, Oncor, USA) for 1.5 h at 37°C. After the end-labeling, sections were incubated with anti-digoxigenin peroxidase for 30 min at room temperature. For the color development, sections were immersed in 3% aminoethylcarbazole for 3 min at room temperature to detect digoxigenin-labeled nuclei. Sections were counterstained by hematoxylin. Three hundred myocyte nuclei were examined in subendo-, mid- and sub-epicardial regions of the risk area, respectively by a microscopy at magnification of 200x. The percentage of TUNEL-positive myocyte nuclei was calculated as %AP.
2.6 Agarose gel electrophoresis of DNA
In a separate series of experiments, transmural myocardial samples (n=5, each) from risk and non-risk areas were frozen in liquid nitrogen and stored at –80°C until use. Myocardial DNA was extracted from 70–80 mg of myocardium by a nucleic acid extraction kit (IsoQuick®, Microprobe, USA). The extracted DNA (10 µg) was loaded on 2% agarose gel containing ethidium bromide and electrophoresed on a flatbed gel apparatus (Mupid-3®, Advance, Japan) at 100 V in TBE buffer (0.04 mol/l Tris, 0.04 mol/l borate acid, 2 mmol/l EDTA, pH 8.0). The gels were photographed under UV light.
2.7 Electron microscopy
In a separate series of experiments, rats were subjected to a 30-min ischemia followed by a 6-h reperfusion with and without DEVD as the previous protocol. Rats were killed and tissue samples (twenty portions per heart, n=2 for each group) were taken: ten from the risk area and ten from the center of non-risk area. These samples were cut into 1-mm cubes and fixed for 4 h at 4°C in 2.5% glutaraldehyde in 0.1 mol/l phosphate buffer. They were postfixed in 1% buffered osmium tetroxide, dehydrated through graded ethanols, and embedded in epoxy resin. Thin sections (80 nm) were cut with a diamond knife and collected on bare 300-mesh nickel grids. They were stained with uranyl acetate and lead citrate, and examined in an electron microscope (Hitachi 700).
2.8 Caspase activity
In a separate series of experiments, rats were subjected to a 30-min ischemia followed by 1- or 4-h reperfusion with and without DEVD (n=3, each). Rats were killed and the myocardium from risk and non-risk area were frozen in liquid nitrogen and stored at –80°C until use. The myocardium was homogenized by a polytron homogenizer and centrifuged at 16 000 g for 20 min. A 20-µl volume of the supernatant was applied for the measurement of caspase activity by CaspACE assay system (Promega, WI, USA) according to the instruction. Briefly, the fluorogenic substrates for caspase-1 and caspase-3 are labeled with the fluorochrome 7-amino-4-methyl coumarin (AMC). The substrates produce a blue fluorescence that can be detected by exposure to UV light at 360 nm. AMC is released from the substrates upon cleavage by caspase-1 or -3. Free AMC produces a yellow-green fluorescence that is measured by a fluorometer at 460 nm. The fluorometric count was normalized by the protein concentration of the supernatant
2.9 Statistical analysis
All values are expressed as mean±SD. Differences in hemodynamic parameters between the groups were analyzed by two-way ANOVA. Fisher's test was used when a significant F value was obtained. Inter-group differences in AR/LV, AN/AR, the percentage of TUNEL-positive nuclei and the caspase activity were analyzed by one-way ANOVA followed by Fisher's test. Differences were considered significant at P<0.05.
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3 Results |
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3.1 Hemodynamic response and arrhythmia
Hemodynamic data are summarized in Table 1. Differences in heart rate, peak LVP and LV(+)dP/dt among the groups were not significant. Ten min after the reperfusion, LVEDP was equally elevated in all three groups. Arrhythmia such as ventricular tachycardia developed in all groups during the ischemic period, especially in the initial 10-min period.
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3.2 Myocytes with tunel-positive nuclei
Fig. 1 shows TUNEL-positive myocyte nuclei photographed at a magnification of 200x. The TUNEL-positive nuclei were mainly observed in the infarcted area, but not in the non-risk area. Fig. 2 shows %AP in each region and its average. The average %AP was reduced from 20±1% to 12±3% by YVAD-CHO and to 10±3% by DEVD-CHO (both P<0.01). TUNEL-positive nuclei were mainly observed in the subendocardial region of the infarcted area. %AP in the subendocardial region was significantly reduced from 45±3% to 25±3% by YVAD-CHO and to 19±4% by DEVD-CHO (both P<0.01). %AP in the mid- and subepicardial regions were not significantly reduced by either caspase inhibitor.
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The number of myocyte nuclei in a microscopic field at a magnification of 200x was counted in the subendocardium of the risk area. Thirty to thirty-five fields were examined in each slice and the counts were averaged (Fig. 3). The number of TUNEL-positive myocyte nuclei in a field was also significantly reduced by YVAD and DEVD, respectively (4.6±1.6 in the vehicle group, 2.3±0.8 in the YVAD group, 1.7±0.5 in the DEVD group, P<0.01 vs. vehicle group, respectively). Conversely, the number of TUNEL-negative myocyte nuclei in a field was significantly increased in DEVD group compared with the vehicle group (P<0.05). However, the total number of myocyte nuclei in a field is identical among the groups.
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3.3 Agarose gel electrophoresis
A series of DNA fragments showing size ranges in multiples of 180–200 bp units is called ladder pattern which indicates apoptotic internucleosomal DNA fragmentation. DNA from the risk areas of the vehicle group showed ladder pattern. The DNA from the risk areas of YVAD and DEVD groups showed obscure ladder pattern (Fig. 4). No ladder pattern was observed in DNA from the non-risk area of all groups.
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3.4 Myocardial infarct size
The size of area at risk was identical among the groups (Fig. 5, left). The infarct size expressed by AN/AR was not significantly different among the groups (Fig. 5, right).
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3.5 Electron microscopic findings
Electron microphotographs of the subendocardial region show that the cytoplasm of myocytes was severely edematous and the myofibrils were disintegrated. The mitochondrias (Mt) were edematous, disrupted, and contained amorphous dense bodies (arrowhead). Red blood cells invading the myocytes were observed, which indicates extensive rupture of the plasma membrane. These findings are equally observed in both vehicle and DEVD groups (Fig. 6).
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3.6 Caspase activity
The caspase activity of the risk area in the vehicle group was significantly increased compared with that of its non-risk area at 4 h after the reperfusion (Fig. 7). However, the caspase activity was not significantly increased at 1 h after the reperfusion. In the DEVD group, the caspase activity of the risk area was significantly suppressed compared with that of the vehicle group (P<0.01, Fig. 7).
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4 Discussion |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionsAcknowledgmentsReferences |
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In the ischemic cell death of myocytes, it has been believed that necrosis is the main feature. Recently, it has been noticed that apoptosis occurs in the ischemia–reperfused myocardium [6,9]. Morphologically, necrosis is characterized by swelling of mitochondrias and the loss of plasma membrane integrity [2]. On the other hand, apoptosis is characterized by chromatin condensation, formation of cell fragments known as ‘apoptotic bodies’, and the absence of inflammation [20]. Biochemically, apoptosis is defined by the internucleosomal degradation of chromosomal DNA into multiples of 180–200 bp on gel electrophoresis termed ladder pattern [1]. In the ischemia–reperfused rabbit hearts, Gottlieb et al. [9] first showed a characteristic feature of apoptosis such as chromatin condensation by electron microscopy and the internucleosomal fragmentation of DNA.
4.1 Effects of caspase inhibitors on infarct size and TUNEL positivity
Viable myocardium can be identified by TTC staining, which identifies NADH amount in mitochondrias [11,19]. This method has been demonstrated to be well correlated with histological quantitation of infarct size [5] and has been widely used to verify cardioprotective effects of various drugs and ischemic preconditioning. In the present study we tested the effect of caspase inhibitors on TTC-defined infarct size. Surprisingly, the infarct size was not reduced by the caspase inhibitors despite a significant reduction in TUNEL-positive myocytes. This contrasts with the effect of ischemic preconditioning which reduces both infarct size and the incidence of DNA fragmentation [15]. The TUNEL method has been widely used to detect apoptosis in various tissues and cell cultures. The detection of ladder pattern and TUNEL positivity has been usually adopted for the marker of apoptosis. In the present study we demonstrated that the incidence of TUNEL-positive myocytes was decreased by the caspase inhibitors. Furthermore, TUNEL-negative myocytes were significantly increased in association with a concomitant reduction in the TUNEL-positive myocytes by the caspase inhibitors. These results support that the caspase inhibitors can reduce damages of DNA. However, the infarct size was not significantly decreased. This raised a question whether the impact of apoptosis on infarct size is small.
4.2 Electron microscopic findings
To further clarify the above question, we examined the electron microscopic features of the ischemic myocardium. Electron microscopic findings showed that the cell membrane integrity was disrupted and the mitochondrial damage was apparent in the subendocardial region where most TUNEL-positive myocytes were observed. These findings were equally observed in both groups administered with vehicle or a caspase inhibitor DEVD. This result strongly suggests that most of the TUNEL positive myocytes have been severely damaged on the cell membrane and mitochondrias, causing TTC negative. Thus it is conceivable to hypothesize that even though the caspase inhibitors successfully inhibit the caspase activation leading to DNA fragmentation, the infarct size is not reduced.
4.3 Effects of DEVD on caspase activity in the ischemic myocardium
During the process of apoptosis, caspases are activated. The inhibition of caspases pharmacologically can block the completion of apoptosis. In the present study we measured the activity of caspase-3 in the risk and non-risk myocardium with and without a caspase inhibitor DEVD. At 4 h after the reperfusion, the caspase-3 activity was significantly increased compared with that in the non-risk area. The augmented caspase-3 activity was suppressed by DEVD. This result indicates that the reduction of TUNEL-positive myocytes can be attributed to the inhibition of caspase-3 activity. Interestingly, the caspase-3 activity was not increased 1 h after the reperfusion, when the ultrastructural damages has already been demonstrated. Considering these results, caspase-3 may be activated after the damages of cell membrane and mitochondrias. If this is true, the caspase inhibitors cannot reduce infarct size even though the DNA fragmentation is attenuated.
Recently, Yaoita et al. [21] demonstrated that a nonspecific broad caspase inhibitor, ZVAD-fmk, administered before the ischemia and every 6 h after reperfusion reduced both infarct size and TUNEL-positive cardiomyocytes 24 h after reperfusion. The discrepancy concerning the infarct size reduction between the studies may be related to the difference of inhibitors and the mode of the drug administration. In particular we did not administer the drugs during the reperfusion period.
TUNEL-positive myocytes were preferentially observed in the subendocardium. The reason for this phenomenon is not clear. Since the ischemic damage is usually more severe in the subendocardium than in the subepicardium, caspase-3 in the subendocardium may be more activated than in the subepicardium. This needs to be further examined.
4.4 Limitations
The method used in the present study is highly specific for detecting the nick of genomic DNA. However, it is possible that the necrotic cells show TUNEL-positive because of a random break in the DNA. However, since the random break of DNA cannot be inhibited by caspase inhibitors, most of the TUNEL positivity in the present study can be attributed to the fragmentation of DNA rather than the random breaking of DNA.
Although DNA from myocytes and non-myocytes is included in the agarose gel electrophoresis, the ladder pattern is not necessarily derived from myocyte fragmentation. However, since most of TUNEL-positive cells originate from myocytes in the present study, the ladder pattern should mainly reflect cardiomyocytes fragmentation.
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5 Conclusions |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionsAcknowledgmentsReferences |
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This study demonstrates that the caspase inhibitors reduce myocyte DNA fragmentation and the caspase activation in the ischemia–reperfused rat heart without reduction in infarct size. The pathophysiological significance of caspase inhibitors in the ischemia–reperfusion injury needs to be evaluated in the chronic stage.
Time for primary review 17 days.
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Acknowledgments |
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TopAbstract1 Introduction2 Methods3 Results4 Discussion5 ConclusionsAcknowledgmentsReferences |
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This study was supported in part by research grants 07670785 from the Ministry of Education, Science and Culture of Japan. We wish to thank Dr. John Ross Jr. for correcting the manuscript. We also thank Miss Rie Ishihara and Miss Kazuko Iwamoto for their technical assistance.
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J. D. McCully, H. Wakiyama, Y.-J. Hsieh, M. Jones, and S. Levitsky Differential contribution of necrosis and apoptosis in myocardial ischemia-reperfusion injury Am J Physiol Heart Circ Physiol, May 1, 2004; 286(5): H1923 - H1935. [Abstract] [Full Text] [PDF]
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 Y. Chandrashekhar, S. Sen, R. Anway, A. Shuros, and I. Anand Long-Term caspase inhibition ameliorates apoptosis, reduces myocardial troponin-I cleavage, protects left ventricular function, and attenuates remodeling in rats with myocardial infarction J. Am. Coll. Cardiol., January 21, 2004; 43(2): 295 - 301. [Abstract] [Full Text] [PDF]
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K. A. Webster and N. H. Bishopric Apoptosis Inhibitors for Heart Disease Circulation, December 16, 2003; 108(24): 2954 - 2956. [Full Text] [PDF]
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Y. Zou, W. Zhu, M. Sakamoto, Y. Qin, H. Akazawa, H. Toko, M. Mizukami, N. Takeda, T. Minamino, H. Takano, et al. Heat Shock Transcription Factor 1 Protects Cardiomyocytes From Ischemia/Reperfusion Injury Circulation, December 16, 2003; 108(24): 3024 - 3030. [Abstract] [Full Text] [PDF]
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 D. M. YELLON and J. M. DOWNEY Preconditioning the Myocardium: From Cellular Physiology to Clinical Cardiology Physiol Rev, October 1, 2003; 83(4): 1113 - 1151. [Abstract] [Full Text] [PDF]
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D. Cai, M. Xaymardan, J. M. Holm, J. Zheng, J. R. Kizer, and J. M. Edelberg Age-associated impairment in TNF-{alpha} cardioprotection from myocardial infarction Am J Physiol Heart Circ Physiol, July 11, 2003; 285(2): H463 - H469. [Abstract] [Full Text] [PDF]
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 G. Li, Y. Xiao, J. L. Estrella, C. A. Ducsay, R. D. Gilbert, and L. Zhang Effect of Fetal Hypoxia on Heart Susceptibility to Ischemia and Reperfusion Injury in the Adult Rat Reproductive Sciences, July 1, 2003; 10(5): 265 - 274. [Abstract] [PDF]
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 P A J Krijnen, R Nijmeijer, C J L M Meijer, C A Visser, C E Hack, and H W M Niessen Apoptosis in myocardial ischaemia and infarction J. Clin. Pathol., November 1, 2002; 55(11): 801 - 811. [Abstract] [Full Text] [PDF]
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 A. Iwata, J. M. Harlan, N. B. Vedder, and R. K. Winn The caspase inhibitor z-VAD is more effective than CD18 adhesion blockade in reducing muscle ischemia-reperfusion injury: implication for clinical trials Blood, August 28, 2002; 100(6): 2077 - 2080. [Abstract] [Full Text] [PDF]
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Z.-Q. Zhao and J. Vinten-Johansen Myocardial apoptosis and ischemic preconditioning Cardiovasc Res, August 15, 2002; 55(3): 438 - 455. [Abstract] [Full Text] [PDF]
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 M. Chen, D.-J. Won, S. Krajewski, and R. A. Gottlieb Calpain and Mitochondria in Ischemia/Reperfusion Injury J. Biol. Chem., August 2, 2002; 277(32): 29181 - 29186. [Abstract] [Full Text] [PDF]
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T. Date, A. J Belanger, S. Mochizuki, J. A Sullivan, L. X Liu, A. Scaria, S. H Cheng, R. J Gregory, and C. Jiang Adenovirus-mediated expression of p35 prevents hypoxia/reoxygenation injury by reducing reactive oxygen species and caspase activity Cardiovasc Res, August 1, 2002; 55(2): 309 - 319. [Abstract] [Full Text] [PDF]
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 C. GILL, R. MESTRIL, and A. SAMALI Losing heart: the role of apoptosis in heart disease--a novel therapeutic target? FASEB J, February 1, 2002; 16(2): 135 - 146. [Abstract] [Full Text] [PDF]
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 C A Dinarello Novel targets for interleukin 18 binding protein Ann Rheum Dis, November 1, 2001; 60(90003): iii18 - 24. [Abstract] [Full Text] [PDF]
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J. Grunenfelder, D. N. Miniati, S. Murata, V. Falk, E. G. Hoyt, M. Kown, M. L. Koransky, and R. C. Robbins Upregulation of Bcl-2 Through Caspase-3 Inhibition Ameliorates Ischemia/Reperfusion Injury in Rat Cardiac Allografts Circulation, September 18, 2001; 104 (2009): I-202 - I-206. [Abstract] [Full Text] [PDF]
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 B. J. Pomerantz, L. L. Reznikov, A. H. Harken, and C. A. Dinarello Inhibition of caspase 1 reduces human myocardial ischemic dysfunction via inhibition of IL-18 and IL-1beta PNAS, February 27, 2001; 98(5): 2871 - 2876. [Abstract] [Full Text] [PDF]
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M. Borgers, L.-M. Voipio-Pulkki, and S. Izumo Apoptosis Cardiovasc Res, February 1, 2000; 45(3): 525 - 527. [Full Text] [PDF]
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J. M. Edelberg, S. H. Lee, M. Kaur, L. Tang, N. M. Feirt, S. McCabe, O. Bramwell, S. C. Wong, and M. K. Hong Platelet-Derived Growth Factor-AB Limits the Extent of Myocardial Infarction in a Rat Model: Feasibility of Restoring Impaired Angiogenic Capacity in the Aging Heart Circulation, February 5, 2002; 105(5): 608 - 613. [Abstract] [Full Text] [PDF]
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