Cardiovascular Research

Cardiovascular Research 2000 45(3):661-670; doi:10.1016/S0008-6363(99)00393-4
© 2000 by European Society of Cardiology

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Copyright © 2000, European Society of Cardiology

Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart

Masanori Nakamura^a, Ning-Ping Wang^a, Zhi-Qing Zhao^a, Josiah N Wilcox^b, Vinod Thourani^a, Robert A Guyton^a and Jakob Vinten-Johansen^a,*

^aDepartment of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA 30365-2225, USA
^bDepartment of Hematology/Oncology, Emory University School of Medicine, Atlanta, GA 30365-2225, USA

^* Corresponding author. Tel.: +1-404-686-2511; fax: +1-404-686-4888 jvinten{at}emory.edu

Received 2 June 1999; accepted 14 October 1999

	Abstract

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Objective: Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study tested the hypothesis that IPC reduces ischemia/reperfusion-induced myocardial apoptosis by inhibiting neutrophil (PMN) accumulation and altering expression of Bcl-2 and Bax proteins. Methods: Eighteen rats were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion with IPC (5 min ischemia and 10 min of reperfusion, n=10) or without IPC (n=8). Myocardial apoptosis was detected histologically using the terminal transferase UTP nick end labeling (TUNEL) assay and confirmed by DNA ladder on agarose gel electrophoresis. PMN accumulation was detected immunohistochemically with anti-rat CD18 antibody (WT3) and expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. Results: IPC significantly decreased TUNEL positive cells (% total nuclei) in the ischemic zone from 28.6±2.8 to 3.4± 0.9 (P<0.05), consistent with the absence of DNA ladders in the IPC group. IPC significantly attenuated PMN accumulation (cells/mm² myocardium) in the ischemic zone from 243±19 to 118±19 (P<0.05). By regression analysis, there was a significant correlation between TUNEL positive cells and accumulated CD18 positive PMNs in the ischemic zone (r=0.8, P<0.001), which was shifted downward by IPC. Densitometrically, IPC significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein in the ischemic zone from 204±57% in the control group to 76±7% (P<0.05), while the expression of Bcl-2 was not different from the non-ischemic zone in either group. Conclusion: These data suggest that ischemic preconditioning may reduce myocardial apoptosis by inhibiting PMN accumulation and down-regulating expression of Bax.

KEYWORDS Apoptosis; Ischemia; Leukocytes; Preconditioning; Reperfusion

	1 Introduction

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
Two distinct types of cell death in myocardium, necrosis and apoptosis, have been linked with ischemia and reperfusion. ATP depletion, cell swelling or cell rupture, loss of cell membrane integrity and a significant inflammatory response characterize the morphologic manifestation of necrosis [1,2]. Apoptosis, on the other hand, usually requires energy and is associated with cell shrinkage, blebbing, and phagocytosis of cell fragments by pedestrian cells without a subsequent inflammatory response [1,3–8]. The hallmark of apoptosis in intact cells is endonucleolytic digestion of nuclei into oligonucleosome sized (180–200 bp) fragments. Although it has been reported that necrotic cell death leads to a destruction of a large group of cells, apoptosis may independently contribute to irreversible myocardial damage [8,9].

Ischemic preconditioning (IPC), as the most potent intervention to reduce a number of pathophysiological consequences of ischemia–reperfusion injury, was first described by Murry et al. [10] in 1986. Since then, studies have shown that IPC attenuates the incidence and severity of reperfusion-induced arrhythmias, prevents endothelial cell dysfunction and reduces infarct size [11–13]. With regard to inhibition of apoptosis by IPC, Gottlieb et al. [14] reported that IPC significantly reduced DNA fragmentation and apoptotic myocyte death in an isolated rabbit heart of 30 min of prolonged metabolic inhibition followed by 4–24 h of recovery. Pilot et al. [15] also showed that IPC significantly attenuated both infarct size and myocardial apoptosis in a rat model of 30 min of ischemia followed by 120 min of reperfusion. However, the potential mechanisms underlying IPC-attenuated myocardial apoptosis after ischemia and reperfusion are not clear at the present time.

Although a large number of genes have been reported to be involved in the regulation of apoptotic cell death, Bcl-2 and the related family member, Bax, play a major role in regulating apoptotic cell death in response to inflammatory stimuli. Evidence in support of this view comes from two studies. A clinical study showed [16] that positive expression of Bcl-2 protein was detected in salvaged myocytes surrounding infarcted tissues while Bax was clearly overexpressed in the infarct area, suggesting that the balance between Bcl-2 and Bax plays a role in salvaging of ischemic tissue. Expression of Bcl-2 protein in the prevention of apoptotic cell death was also demonstrated in the isolated rat myocyte [17]. In addition, reactive oxygen substances released from the activated neutrophil (PMN) have been reported to be involved in the induction of apoptosis [18]. Accordingly, the purpose of this study was to test the hypothesis that IPC attenuates the extent of ischemia/reperfusion-induced apoptosis by regulating expression of the Bcl-2 family and inhibiting PMN activation and accumulation.

	2 Methods

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
All animals received human care in compliance with the ‘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 1996).

2.1 Surgical preparation
Male rats weighing 250–300 g were anesthetized intraperitoneally with sodium pentobarbital (60 mg/kg) followed by continuous inhalation of 1.5% isoflurane after endotracheal intubation with a 14 gauge tube. The animals were ventilated with 100% oxygen using a rodent respirator (HARVARD Rodent Ventilator Model 683, 70 breaths per minute). Normal blood gas levels and acid–base status were maintained by adjustment of the rate and tidal volume or by intravenous administration of sodium bicarbonate as necessary. The left carotid artery and external jugular vein were cannulated with a French micromanometer pressure transducer (MPC-500, Millar Instruments, Houston, Texas) to monitor mean arterial pressure and heart rate. The chest was opened through the fourth intercostal space on the left side of the chest, and the ribs were gently retracted to expose the heart. After pericardiotomy, A 6-0 prolene (Ethicon, NJ) ligature was placed under the left main coronary artery and the ends of the tie were threaded through a small plastic (PE50) tube to form a snare for reversible left coronary artery (LCA) occlusion. Heparinization was maintained during the experimental period with a bolus injection of 100 U/kg of sodium heparin. The body temperature was monitored by rectal thermometer and maintained constant between 37 and 38°C by heating pads.

2.2 Experimental protocol
After a period of post-surgical stabilization, rats were randomly assigned to one of two groups. Control group (n=8): 30 min of LCA occlusion followed by 180 min of reperfusion. IPC group (n=10): Preconditioning protocol consisted of one cycle of 5 min of ischemia and 10 min of reperfusion prior to sustained 30 min of LCA occlusion followed by 180 min of reperfusion. At the end of the experiment, the LCA was re-occluded and 1 ml of 20% Unisperse blue dye was injected into the external jugular vein and circulated for 1 min to outline the normal zone and area at risk. The ischemic and necrotic zones were separated by triphenyltetrazolium chloride staining [19]. Tissue samples from the normal and ischemic zones were used to quantify tissue DNA laddering, myocardial apoptosis, PMN accumulation by immunohistochemistry and expression of Bcl-2 and Bax proteins (see below).

2.3 Tissue preparation
The left ventricular tissue samples from non-ischemic and ischemic zones were placed in tubes for DNA isolation and Western blot assay, and embedded in optimal cutting temperature compound (O.C.T., Sakura Finetek), then frozen in liquid nitrogen and stored at –70°C for detection of apoptotic cells using the TUNEL method or for detection of PMN accumulation by immunohistochemistry. Cryosections (7 µm thick) were obtained using a Hacker–Bright cryostat and thaw-mounted onto Vectabond (Vector Laboratories, Burlingame, CA) coated slides or Fisher-Plus (Fisher Scientific) slides, refrozen, and stored at –70°C with desiccant until use.

2.4 The presence of DNA ladder in agarose gel electrophoresis
Frozen tissue samples (20–30 mg each) from non-ischemic and ischemic zones were minced in 600 µl of lysis buffer (Puregene DNA Isolation Kit) and were quickly homogenized using 30–50 strokes with a microfuge tube pestle. The tissue was digested with 100 µg/ml of proteinase K (Sigma) at 56°C for 3–4 h and incubated with RNase A at 37°C for 1 h. After incubation, tissues were precipitated and centrifuged at 16 000xg for 5 min. Supernatants containing DNA were precipitated with isopropanol. After centrifugation at 16 000xg for 5 min, the resulting DNA pellets were washed with 75% ethanol and dissolved in DNA hydration solution at 260 nm by spectrophotometry. A 10 µg amount of DNA was loaded onto 1.5% agarose gels containing 0.5 µg/ml ethidium bromide. DNA electrophoresis was carried out at 80 V for 1 to 2 h. DNA ladders, an indicator of tissue apoptotic nucleosomal DNA fragmentation, were visualized under ultraviolet light and photographed for permanent records.

2.5 The detection of myocardial apoptosis by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-X nick end labeling (TUNEL)
Frozen tissue samples of non-ischemic and ischemic zones were cut into 7 µm sections, fixed in 4% paraformaldehyde in PBS for 20 min at room temperature, and incubated with proteinase K (1 µg/ml) in PBS for 20 min. DNA fragments in the tissue sections were determined using an in situ cell death detection kit (Boehringer Mannheim, Ridgefield, CT). Briefly, the enzyme, terminal deoxynucleotidyl transferase (TdT), was used to incorporate digoxigenin-conjugated dUTP to the 3'-end of single- and double-stranded DNA (DNA fragments). The TUNEL signal was then detected by an anti-fluorescein antibody conjugated with alkaline phosphatase, a reporter enzyme that catalytically generates a red-colored product from Vector Red substrate. The slides were counterstained with hematoxylin, dehydrated in graded alcohols and covered with Accumount medium. For each slide, color video images of ten separate fields were captured randomly and digitized by using a x20 objective on an Olympus IX50 microscope connected to an IBM computer. The cells with clear nuclear labeling were defined as TUNEL positive cells. The apoptotic cells were calculated as percentage of TUNEL positive cells using the following formula: number of TUNEL positive cell nuclei/(number of TUNEL positive cell nuclei+number of total cell nuclei)x100.

2.6 Western blot analysis of Bcl-2 and Bax proteins
Tissue samples from non-ischemic and ischemic zones were used in determining the expression of Bcl-2 and Bax proteins. Tissue samples (100 mg) for each anatomical zone were homogenized with 500 µl lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate: 0.1% SDS in 1x PBS) at 4°C for 20 s, incubated on ice for 2–3 h, and then spun down twice at 16 000xg for 20 min. Protein concentration was measured by the DC Protein Assay (Bio Rad). An 80 µg amount of total protein for each lane was mixed with loading buffer (5% beta mercaptoethanol, 0.05% bromophenol blue, 75 mM Tris–HCl, pH 6.8, 2% SDS and 10% Glycerol), boiled for 4 min, and loaded onto a 4–15% gradient SDS–polyacrylamide gel using Mini Protean II Dual Stab Cell (Bio Rad). Proteins were transferred on nitrocellulose filter in the presence of a glycine/methanol transfer buffer (20 mM Tris base, 0.15 M glycine, 20% methanol) in Mini Protean II Transfer system (Bio Rad). The nitrocellulose filter was blocked with 6% milk in 1x TBS–T buffer (20 mM Tris–HCl pH 7.6, 137 mM NaCl, 0.05% Tween-20) for 1 h at room temperature. Membranes were subsequently exposed to rabbit polyclonal anti-rat Bcl-2 and rabbit polyclonal anti-rat Bax (Pharmingen, San Diego) at 1:1000 concentration in 6% milk in TBS–T for 1 h, respectively. Bound antibody was detected by horseradish peroxidase conjugated anti-rabbit IgG. Finally, ECL detection reagents were employed to visualize the peroxidase reaction product (Amersham). The Bcl-2 protein was detected as a 26-kDa band and the Bax protein was detected as a 21-kDa band. Monoclonal anti-actin antibody (Sigma) was used as internal control. The scanned image was imported into Adobe Photoshop software, the density at specific molecular weights was measured by NIH image analysis software on a Power Macintosh. In comparison between the groups, data were shown as % density of bands vs. non-ischemic tissue.

2.7 The detection of PMN accumulation with immunohistochemistry
Cryostat sections of 7 µm thickness were prepared. After fixing in acetone and air drying, the sections were blocked for endogenous peroxidase with 3% H₂O₂/methanol, and then treated with 1% gelatin in PBS for blocking non-specific binding. The sections were incubated with monoclonal anti-rat CD18 antibody (WT3) for 1–2 h. The slides were then washed in PBS, and incubated with a 1/400 dilution of biotinylated horse anti-mouse IgG (Vector Laboratories). The sections were stained using the ABC-peroxidase kit (Vector Laboratories) and substrated with 3,3'-diaminobenzidine tetrahydrochloride (Sigma). The slides were dehydrated in graded alcohols and Americlear, and coverslipped with hematoxylin counterstaining. Single- and double-label immunohistochemistry experiments were controlled by either elimination of the primary antibody or incubation of the tissue with a non-immune IgG. PMN accumulation was determined as the number of CD18 positive cells per square millimeter of tissue. To distinguish whether apoptotic cells were PMNs, double immunohistochemical analysis was performed by staining first with TUNEL followed by anti-CD18 antibody for PMNs.

2.8 Statistical analysis
All values are expressed as mean±standard errors (SEM). Comparisons between groups were assessed by one way analysis of variance (ANOVA) followed by Bonferroni's post-hoc test. Hemodynamic data between the groups were analyzed by t-test at each time point. A P value <0.05 was considered significant.

	3 Results

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
3.1 Hemodynamic data
Hemodynamic data for mean aortic pressure, heart rate, rate pressure product in both groups are shown in Fig. 1. There were no significant differences in any measured variables between the two groups at baseline. Although IPC reduced mean aortic pressure during 5 min of coronary occlusion, it returned to control values at the end of 10 min of reperfusion. There was also no group difference in variables at each time point during 30 min of ischemia and 180 min of reperfusion.

View larger version (10K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Hemodynamic data at baseline, ischemic preconditioning (IPC) and reperfusion. MAP, mean aortic pressure; HR, heart rate; RPP, rate-pressure product. MAP was significantly decreased during IPC. There were no statistically significant differences between experimental groups for HR or RPP at any time points. Values are means±SEM. *P<0.05 vs. control group (I/R).

 
3.2 DNA fragmentation after ischemia and reperfusion
Myocardial DNA fragmentation in non-ischemic and ischemic zones is illustrated in Fig. 2. No visible ‘ladders’ were shown in the non-ischemic zone in either group after 30 min ischemia and 180 min of reperfusion. In contrast, genomic DNA isolated from the ischemic zone in the control group produced a typical ‘ladder’ pattern from all eight rats, indicating DNA nucleosomal fragmentation after ischemia and reperfusion. IPC significantly reduced the appearance of DNA ‘ladders’ in the ischemic zone. Among ten animals, six of them showed little DNA laddering, as indicated in lane 8 on Fig. 2, the remaining four animals did not show any DNA ladder pattern (indicated in lane 6 in Fig. 2). A ‘smear’ background in the DNA ladder pattern, which has been suggested to indicate random DNA fragmentation involved in necrotic cell death, was not found in either group.

View larger version (39K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Detection of DNA fragmentation using agarose gel electrophoresis. Numbers at the bottom of the figure indicate that the non-ischemic zone (lanes 1 and 3 in the control group and lanes 5 and 7 in the IPC group) and ischemic zone (lanes 2 and 4 in the control group and lanes 6 and 8 in the IPC group). No ladders were detected in the non-ischemic zone. IPC significantly decreased the intensity of appearance of the DNA ladder in the ischemic zone compared with the control group.

 
3.3 Apoptotic cell death after ischemia and reperfusion
To exclude the possibility that the absence of apoptotic cells in the non-ischemic zone by TUNEL staining was due to interference by Unisperse blue pigment, we treated tissue sections using RNase A in the non-ischemic zone with and without blue dye, and found that both of these sections showed a similar density of positive nuclei after TUNEL staining. Therefore, the presence of Unisperse blue pigment did not interface with TUNEL staining. TUNEL positive nuclei were not found in the non-ischemic zone after ischemia and reperfusion in both groups. However, ischemia/reperfusion increased the number of TUNEL positive cells expressed as the percent of total nuclei in the ischemic zone by 28.6±1.1% compared to control hearts (Fig. 3A), consistent with the appearance of DNA fragmentation in these zones. One cycle of IPC significantly decreased the number of TUNEL positive cells by 3.4±0.9% (P<0.05 vs. control group, Fig. 3B).

View larger version (120K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Detection of apoptotic myocytes in the ischemic zone in the control group (A) and IPC group (B) using the terminal transferase dUTP nick end labeling (TUNEL) technique. Red staining indicates TUNEL positive cells. IPC significantly reduced the number of TUNEL positive cells vs. control group after ischemia and reperfusion. Bar indicates 0.05 mm in length.

 
3.4 PMN accumulation after ischemia and reperfusion
PMN accumulation in the ischemic myocardium in both groups was detected by immunohistochemistry with monoclonal anti-rat PMN CD18 antibody, WT3. In both groups, few CD18 positive cells (cells/mm² myocardium) were found in the non-ischemic tissue sections. In contrast, the number of CD18 positive cells in the ischemic zone in the control group was significantly increased to 243±19 (Fig. 4A), compared with 16±4 in the non-ischemic zone, P<0.05. The increased number of CD18 positive cells was significantly attenuated by 44% in the IPC group (Fig. 4B, 118±19 vs. control group, P<0.05).

View larger version (98K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Localization of neutrophil (PMN) in the ischemic zone in the control group (A) and IPC group (B) using immunohistochemistry with anti-CD18 antibody (WT3). Brown staining indicates CD18 positive cells. IPC significantly inhibited PMN accumulation after ischemia and reperfusion vs. control group. Bar indicates 0.05 mm in length.

 
3.5 The correlation between accumulated PMNs and apoptotic myocytes
We assessed CD18 positive cells and TUNEL positive cells in the tissue section removed from the ischemic zones in both groups. There was a significant linear relationship (r=0.8, P<0.001) between CD18 positive cells and TUNEL positive cells, suggesting that PMN accumulation may be associated with the development of myocardial apoptosis. To exclude the possibility that TUNEL positive cells include accumulated PMNs, immunohistochemical double staining with TUNEL and anti-CD18 was applied on the same tissue slide. Few CD18 positive cells were stained by the TUNEL method (Fig. 5), suggesting that TUNEL positive cells are mainly myocytes.

View larger version (96K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Double staining using the terminal transferase dUTP nick end labeling (TUNEL) technique and immunohistochemistry with anti-CD18 antibody on the same tissue slide after ischemia and reperfusion. Brown staining (arrow head) indicates PMNs and red staining (arrow) indicates apoptotic myocytes, respectively. An absence of TUNEL positive PMN suggests that apoptotic cells are not PMNs. Figures are representative of at least four separate experiments. Bar indicates 0.1 mm in length.

 
3.6 Expression of Bcl-2 and Bax proteins after ischemia and reperfusion
The changes in expression of Bcl-2 and Bax proteins were analyzed by Western blot assay (Fig. 6). Bcl-2 and Bax were present in the non-ischemic zone in both groups (Fig. 6A). Densitometrically, there was no significant difference between the groups in expression of the Bcl-2 protein after ischemia and reperfusion (104±35% in the control group vs. 96±32% in the IPC group). The Bax protein (Fig. 6B), however, was significantly increased in the control group by 204±57% compared with its expression in the non-ischemic zone (P<0.05). In contrast, the expression of Bax protein was significantly attenuated in the IPC group (76±7% vs. control group, P<0.05), suggesting that IPC may reduce myocardial apoptosis by inhibiting expression of Bax protein in the ischemic zone.

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Expression of Bcl-2 and Bax proteins was visualized by Western blot analysis. Protein lysates (80 µg) for each lane in the non-ischemic and ischemic zones from control (I/R) and IPC groups were loaded on a SDS–polyacrylamide gel. Densitometrically, there was no significant difference between the groups in expression of Bcl-2 protein (A). However, overexpressed Bax protein was significantly attenuated in the IPC group (B). Immunodetection of actin (top) with a monoclonal anti-actin antibody was performed as an internal control. Figures are representative of at least six separate experiments.

 

	4 Discussion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
The present study demonstrates that apoptosis confirmed by in situ TUNEL labeling and DNA fragmentation was significantly present in the myocardium after a brief period of ischemia followed by reperfusion. The positive correlation between neutrophil accumulation and the TUNEL positive cells, as well as overexpressed Bax protein, suggested a possible role for neutrophils and Bax protein in the pathogenesis of myocardial apoptosis triggered by ischemia and reperfusion. Ischemic preconditioning was associated with a decrease in expression of Bax protein and reduced neutrophil accumulation, this may partially explain, therefore, the protective effect of ischemic preconditioning after ischemia and reperfusion.

The internucleosomal DNA fragmentation observed in agarose gels and histochemical visualization of nuclei by TUNEL staining has been widely used to define myocardial apoptosis. However, there are some recent reports questioning whether apoptotic cells after ischemia and reperfusion detected by the TUNEL staining may also include necrotic cells. [20,21] In the present study, we analyzed myocardial apoptosis in the ischemic zone from a model of 30 min of ischemia followed by 180 min of reperfusion, which may have included necrotic tissue. Without analysis by electron microscopy, we can not exclude the possibility that the TUNEL positive cells contained necrotic cells. However, a recent report from our laboratory by Zhao et al. [22] showed that the TUNEL positive cells were not clearly detected in the tissue sections obtained from necrotic myocardium in dogs subjected to 7 h of ischemia without reperfusion, suggesting that the TUNEL method may be more specific for staining of apoptotic cells [22]. In the present study, the lack of a diffuse smear pattern, an indicator of random DNA breakdown occurring with necrosis, observed on gel electrophoresis, support that the TUNEL positive cells are predominantly apoptotic cells, rather than necrotic cells. In addition, in order to exclude the possibility that neutrophils accumulated in ischemic myocardium after reperfusion may undergo apoptosis, and therefore contribute to TUNEL positive cells, we performed immunohistochemical double staining on the same tissue slide with anti-neutrophil antibody and the TUNEL method. The absence of TUNEL positive neutrophils in these tissue sections suggested that tissue DNA fragmentation on electrophoresis gels after ischemia and reperfusion was associated mainly with myocyte DNA fragmentation. These data are consistent with the absence of apoptotic neutrophils in other species such as cat, dog and human after ischemia and reperfusion observed by other investigators [22–24].

Apoptosis, as a distinct type of cell death, is governed by a number of regulating genes mediated by apoptotic signals. One of the best characterized pathways for the initiation and regulation of apoptosis involves the binding of extracellular death signal proteins (e.g. TNFa and FasL) to their cell surface receptors, the activation of the intracellular execution step (e.g. apoptosis-associated caspase), and down or up-regulation of the Bcl-2 family. Members of the Bcl-2 family that act as inhibitors of apoptosis include Bcl-2, Bcl-X_L and Bcl-w, and those that act like promoters of apoptosis include Bax, Bad, Bak and Bcl-Xs. It is suggested that protein–protein interactions between Bcl-2 family members are a major player in controlling the apoptotic process [4,8,25–27]. For example, Bax may form homodimers to accelerate cell death, or heterodimers with either Bcl-2 and Bcl-X_L to inhibit cell death. Therefore, a change in the ratio of Bcl-2 and Bax protein expression may attenuate the anti-apoptotic effect of Bcl-2 in reducing post-ischemic myocyte apoptosis [5,8]. Furthermore, the induction of the Bax homodimer has been reported to trigger a signaling process of apoptotic death independently. [28] Although ischemia and reperfusion did not alter Bcl-2 expression per se in post-ischemic myocardium in the present study, overexpression of Bax may change the ratio of Bcl-2 and Bax, thereby promoting cell death. An anti-apoptotic effect of Bcl-2 in salvaging ischemic myocyte and proapoptotic effect of Bax in promoting apoptotic cell death have been reported in clinical and experimental studies [16,17].

The role of neutrophils in ischemia/reperfusion-induced necrotic myocyte death has been extensively investigated in the past years [29–31]. The effect of reactive oxygen species and proteases released from activated neutrophils on promoting apoptosis has been documented recently [3,18,32]. However, the precise effect of neutrophils on the pathogenesis of myocardial apoptosis is not clear. Gottlieb et al. showed that granulocytopenia did not prevent ischemia/reperfusion-induced myocardial apoptosis in rabbits [3], arguing against the direct involvement of neutrophils, while Fliss et al. found that accumulated neutrophils in ischemic myocardium were associated with development of apoptotic cell death in rats [32]. As shown in the present study, TUNEL positive cells are significantly correlated with the numbers of accumulated neutrophils. This observation is in agreement with a previous report in which accumulated neutrophils after ischemia and reperfusion are positively correlated with the development of myocardial apoptosis in dog [22]. However, it is still not clear whether the presence of neutrophils plays a causative role in the pathogenesis of apoptosis.

Ischemic preconditioning, as an endogenous cardioprotective adaptation of the heart to a prolonged ischemia/reperfusion injury, has been demonstrated in all species. In addition to the universal observation of limiting necrotic cell death, ischemic preconditioning has been found in some, but not all studies, to ameliorate post-ischemic regional contractile dysfunction, ischemic contracture and arrhythmias [13,33,34]. Moreover, recent evidence suggests that the beneficial effect of ischemic preconditioning may also extend to reducing neutrophil adherence to ischemic/reperfused coronary artery endothelium, attenuating neutrophil-mediated coronary artery endothelial dysfunction, and further limiting neutrophil accumulation in ischemic myocardium after reperfusion [35–37]. However, the specific mechanisms underlying the attenuation of apoptotic cell death after ischemia and reperfusion related to neutrophil function by ischemic preconditioning are still unclear. In the present study, we observed a linear correlation between neutrophil accumulation and the amount of apoptotic cell death after ischemia and reperfusion. Studies have shown that neutrophils may be involved in the pathogenesis of apoptosis by releasing various cytokines and oxidant species [38–41]. Reactive oxygen species such as superoxide and hydroxyl radicals are suggested as the predominant mediators of reperfusion-induced necrotic cell death. Interestingly, many of the agents commonly used to induce apoptosis also induce oxidative stress in the cell. A number of antioxidants and radical scavengers have been shown to inhibit characteristic features of apoptotic cell death [18,42,43]. A recent report indicated that in a canine model of 90 min of ischemia followed by 48 h of reperfusion bolus injection of recombinant human superoxide dismutase, an oxygen radical scavenger, followed by continuous infusion over 60 min of reperfusion significantly reduced TUNEL labeled positive cells [41]. In the present study, ischemic preconditioning inhibited neutrophil accumulation in the ischemic–reperfused myocardium and thereby would decrease the apoptotic cells triggered by neutrophil-derived cytokines and oxidant species. Furthermore, ischemic preconditioning attenuated the expression of Bax protein in ischemic myocardium, and this may help increase the ratio of Bcl-2 and Bax proteins in preventing the progression of apoptosis. Recently, Baghelai et al. reported that attenuation of apoptosis by ₁-adrenoceptor activation-mediated delayed preconditioning was associated with an inhibition in expression of Bax protein in an isolated rabbit heart of 45 min of global ischemia and 120 min of reperfusion, suggesting a role of Bcl_x/Bax ratio in limiting apoptotic cell death [44].

Alternative pathways have been identified, which trigger apoptosis independent of neutrophils and gene expression. Gottlieb et al. [14] found in an isolated rabbit myocyte preparation that apoptosis could be prevented by ischemic preconditioning through activation of protein kinase C to diminish intracellular acidification, suggesting that intracellular acidosis may be involved in inducing apoptotic cell death. Supporting this point of view of ischemic preconditioning-mediated protection from apoptosis, other studies have also shown that a reduction of intracellular acidosis via protein kinase C activation is associated with ischemic preconditioning in in vivo rat hearts. [32,45,46]

	5 Conclusion

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 
The present study used a rat model of transient coronary occlusion to evaluate the effect of ischemic preconditioning on myocardial apoptosis. These data suggest that accumulated neutrophils and upregulation of Bax protein in ischemic myocardium may be involved in the developing ischemia/reperfusion-induced myocardial apoptosis, and ischemic preconditioning may reduce apoptotic cell death in part by inhibiting PMN accumulation and downregulating the expression of Bax protein in ischemic myocardium.

Time for primary review 29 days.

	Acknowledgements

 
The authors are grateful for the technical contributions of Jill Robinson and Sara Katzmark in performing this study, and for the assistance of Gail H. Nechtman in preparing the manuscript. This study was supported by grants from the National American Heart Association (Scientist Development Award, Z.-Q. Zhao) and (Grant-In-Aid, J. Vinten-Johansen) as well as the Carlyle Fraser Heart Center of Emory University.

	References

Top Abstract 1 Introduction 2 Methods 3 Results 4 Discussion 5 Conclusion References

 

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