Journal Logo

Experimental Transplantation

Amelioration of myocardial global ischemia/reperfusion injury with volume-regulatory chloride channel inhibitors in vivo1

Mizoguchi, Kazuhiro2; Maeta, Hajime2; Yamamoto, Akira3; Oe, Masahiro2; Kosaka, Hiroaki3,4

Author Information

Abstract

Cardioprotection is an important issue in cardiac surgery, especially in cardiac transplantation. In earlier concepts, the cell death occurring after ischemia/reperfusion (I/R) in the heart was largely attributed to necrosis, the area of which is determined by triphenyltetrazolium chloride (TTC) staining. Cardiomyocyte apoptosis has been recently observed during I/R (1–3), particularly in the border zone around the necrotic core in a regionally infarcted heart with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method (4,5). Global I/R also induce cardiomyocyte apoptosis in vivo (6,7). However, the contribution of pure apoptosis to myocyte loss during both myocardial ischemia and reperfusion has been questioned by many investigators (8,9), and Gottlieb and Engler (10) proposed that it is more useful to view cell death in the context of whether it can be prevented.

Although apoptosis is accompanied by normotonic shrinkage of cells, it was recently reported that if volume-regulatory Cl channels are blocked, cell shrinkage and subsequent apoptotic events are not induced on the addition of pro-apoptotic reagents in hematopoietic cells and immortalized cell lines (11,12). We thus speculate that volume-regulatory Cl channel inhibitors can prevent cardiomyocyte apoptosis induced by I/R, because caspase inhibitors have been demonstrated to prevent cardiomyocyte apoptosis induced by I/R in vivo (5,13,14).

In this study, we construct a model of global ischemia, followed by reperfusion via heterotopic transplantation into the abdominal aortae of recipient rats. This model should be useful for simulating the cardioprotection on cardiac transplantation. With this model, we examined whether volume-regulatory Cl channel inhibitors can prevent cardiac damage to a similar extent to a broad-spectrum caspase inhibitor, by means of TUNEL staining, genomic DNA electrophoresis, caspase-3-like activity assaying, TTC staining, and ultrasound cardiography. For further characterization of apoptotic, necrotic, or viable cardiomyocytes in this model, horseradish peroxidase (HRP) infiltration assaying, TTC staining, and TUNEL staining were simultaneously performed for one frozen section.

MATERIALS AND METHODS

All experiments were performed in accordance with the Guidelines for the Care and Use of Laboratory Animals (NIH Publication No.85-23, revised 1996) and were approved by the Animal Research Committee of Kagawa Medical University.

Global Ischemia/Reperfusion Model

For this study, 247, 7-week-old, male Wistar rats (180–220 g) were used (Table 1). The rats were anesthetized through inhalation of 1–2% isoflurane, and heterotopic heart transplantation was performed by the modified technique of Ono and Lindsey (15). Briefly, after heparinization (1000 U/kg), the donor hearts were arrested with a cardioplegic solution (ice-cold 5% glucose solution containing 100 mEq/l of KCl and 50 U/ml of heparin) and harvested within 8 min (Fig. 1A). In the warm (n=6) and cold (n=6) I/R groups, the excised hearts were incubated for 8 min in lactated Ringer’s solution containing 50 U/ml of heparin at 37°C and 4°C, respectively. After the warm or cold ischemia, the hearts were stored in the cardioplegic solution. The donor hearts were implanted into the abdominal aortae and inferior vena cavae of recipient rats with aorto-aortic anastomosis and pulmonary-cava anastomosis. Throughout the transplantation procedure (50 min), the donor hearts were covered with ice slush.

Table 1
Table 1:
The number of rats for each protocol
Figure 1
Figure 1:
Protocols for global ischemia/reperfusion (I/R) via heterotopic heart transplantation into the abdominal aortae of recipient rats. (A) Protocols for the warm and cold I/R groups, which were the same except for the temperature during ischemia, i.e., 37°C and 4°C, respectively. (B) Protocol for the effects of a broad-spectrum caspase inhibitor and volume-regulatory chloride channel inhibitors on global I/R injury.

At 2, 4, 8, 14, 24, or 48 hr after reperfusion, the transplanted hearts were excised, perfused with lactated Ringer’s solution containing heparin, and then sectioned transversely into two specimens. The apical side of each specimen was fixed in phosphate-buffered saline (PBS) (pH 7.4) containing 4% paraformaldehyde (Sigma Chemical Co., St. Louis, MO). The basal side of the specimen was used to extract genomic DNA.

Effects of a Broad-spectrum Caspase Inhibitor and Volume-Regulatory Cl Channel Inhibitors

We examined the in vivo effects of a broad-spectrum caspase inhibitor (16), benzoyloxycarbonyl-Asp-CH2OC(O)-2,6-dichlorobenzene (Z-Asp-DCB; Peptide Institute, Osaka, Japan), and two volume-regulatory Cl channel inhibitors (12), 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS; Sigma) and 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB; Sigma), on the global ischemia-reperfused hearts (Fig. 1B). PBS (460 μl), containing 3.5 mg/kg of Z-Asp-DCB (n=6), 28 mg/kg of DIDS (n=6), or 15 mg/kg of NPPB (n=6), dissolved in 140 μl of dimethylsulfoxide (DMSO; Sigma) was administered twice intravenously over 5 min, first to the donor rat 30 min before cardioplegic arrest and second to the recipient rat just after reperfusion. A vehicle group (n=7) was also prepared. At 24 hr after reperfusion, the transplanted hearts were removed for TUNEL staining, genomic DNA extraction, and TTC staining after determining the fractional shortening.

TUNEL Staining

After fixation with 4% paraformaldehyde, each heart specimen was embedded in a paraffin block and cut transversely (4-μm thick). TUNEL staining was performed with a Dead End Colorimetric Detection System (Promega Corporation, Madison, WI). Labeled DNA was detected with streptavidin-peroxidase and developed with 3,3′-diaminobenzidine (DAB; Vector Laboratories, Burlingame, CA) and H2O2. Counterstaining was performed with hematoxylin. TUNEL-positive and negative nuclei were stained dark brown and light violet, respectively. The left ventricular wall was separated into four regions: anterior, posterior, lateral, and septal wall. Areas of the four regions were searched by means of orderly shifting of the visual field using the outer grids of the eyepiece for orientation, as described previously (5). The number of myocyte nuclei in microscopic fields (×400) determined per one slide was 3442±121 in 79.3±0.96 fields. TUNEL-positivity was expressed as the percentage of TUNEL-positive cardiomyocyte nuclei among the total cardiomyocyte nuclei.

Cardiomyocyte nuclei have an elliptical shape in longitudinal sections and are surrounded by myofibers. TUNEL-positive cardiomyocyte nuclei were distinguishable from TUNEL-positive noncardiomyocyte nuclei, such as those of fibroblasts and infiltrated inflammatory cells, which were scarce in all fields.

DNA Fragmentation

Extraction of genomic DNA from each left ventricular specimen was performed by the spin column procedure (QIAamp Tissue Kit; Qiagen, Hilden, Germany). The extracted DNA (7.5 μg) was analyzed by electrophoresis on an agarose gel (1.8%) containing ethidium bromide (1 μg/ml). One hundred base pair DNA ladder (New England Biolabs, Beverly, MA) was used as molecular weight standards.

Caspase-3-like Activity

Caspase-3-like activity was assayed with extracts of the transplanted hearts in four groups, i.e., warm I/R (4 hr), + Z-Asp-DCB (4 hr), + DIDS (4 hr), and + NPPB (4 hr) groups, which had undergone 8 min of warm global ischemia followed by 4 hr of reperfusion (n=3 each). Tissue samples were homogenized using glass beads in 1 ml of lysis buffer comprising 25 mM HEPES/NaOH (pH 7.5), 5 mM MgCl2, 5 mM EDTA, 1 mM EGTA, 2 mM PMSF, 5 mM DTT, 10% sucrose, 10% CHAPS, 0.1% NP40, 10 μg/ml pepstatin, 10 μg/ml leupeptin, and 10 μg/ml aprotinin. The homogenates were centrifuged at 19,000 g for 30 min at 4°C. Caspase-3-like activity in the supernatants was assayed fluorometrically, using 7-amino-4-methylcoumarin (AMC)-DEVD, as described in the manufacturer’s instructions for a commercially available kit (CaspACE Assay System; Promega). The levels of released AMC were measured with a multiplate fluorescence reader (FLx800; BIO-TEK INSTRUMENTS, Winooski, VT) with excitation at 380 nm and emission at 460 nm after ultrafiltration of the reaction mixture with molecular cut-off filters (Microcon Centrifugal Filter Device YM-3; Millipore, Bedford, MA). The caspase-3-like activity was defined as the amount of AMC per min per μg protein and expressed as a percentage of the activity of control hearts (without I/R). Protein concentrations were determined by means of the Bradford assay (Bio-Rad, Richmond, CA). The caspase-3-like activity levels in control hearts and after 8-min warm ischemia followed by 4-hr reperfusion were 899.6±81.7 and 2092.5±180.9 (pmol/min/μg protein), respectively.

Assessment of the Dynamic Function of Global Ischemia-reperfused Hearts

At 24 hr after reperfusion, recipient rats were anesthetized through inhalation of 1–2% isoflurane. The rats were placed in the supine position, and then fractional shortening of the left ventricles in the transplanted hearts was measured through the abdominal wall, using ultrasound cardiography at a frequency of 7.5 MHz (SSD-2200; Aloka, Tokyo, Japan). These measurements were performed at the level of the papillary muscle of the transplanted hearts. Fractional shortening (FS) was calculated as:

FS (%)=100×([left ventricular diameter at the end diastolic phase] − [left ventricular diameter at the end systolic phase])/(left ventricular diameter at the end diastolic phase).

TTC Staining

To determine the cardioprotective effects of inhibitors, we performed TTC staining (17) 24 hr after reperfusion in the four groups, i.e., warm I/R (24 hr), + Z-Asp-DCB (24 hr), + DIDS (24 hr), and + NPPB (24 hr) groups. After heparinization, the transplanted hearts were excised, perfused with lactated Ringer’s solution, and then sectioned transversely into two specimens. The basal sides of the specimens were incubated with 2% TTC (Sigma) in PBS for 30 min at 37°C. The scanned images of the TTC-stained heart sections were analyzed using NIH image software for planimetry. The TTC-unstained area was expressed as a percentage of the total left ventricle area.

Simultaneous HRP Infiltration Assaying, TTC Staining, and TUNEL Staining

To assess apoptotic, necrotic, viable, or reversibly injured regions in the present model, HRP (Type II, Sigma) infiltration assaying, TTC staining, and TUNEL staining were simultaneously performed according to the reports by Takashi et al. (18) and Takashi and Ashraf (19). Briefly, after 8 min of warm ischemia followed by 24 hr of reperfusion, 100 mg/kg of HRP in 500 μl of PBS was administered intravenously over 5 min before removing the transplanted hearts to assess membrane rupture or permeability. Then, the removed hearts were perfused with lactated Ringer’s solution containing heparin via a Langendorff apparatus for 2 min, perfused with a TTC solution at 37°C for 5 min, perfusion-fixed with 4% paraformaldehyde in PBS for 10 min, frozen in an optimum cutting temperature (OCT) compound (Tissue-Tek; Sakura Finetechnical, Tokyo, Japan) in liquid nitrogen, and then cut into 10-μm thick sections. First, to investigate alterations in sarcolemmal integrity and permeability, the sections were washed with PBS and then incubated for 10 min at room temperature in PBS containing H2O2 and Vector SG (Vector Laboratories) chromogen, a substrate for HRP. HRP-positivity was indicated by a green color. Second, after blocking of the activity of injected HRP with 3% H2O2 for 3 min, TUNEL staining was performed. TUNEL-positive nuclei were stained brown with DAB.

Statistical Analysis

All data are mean±SE. All statistical comparisons were performed using a commercially available statistical software package for Macintosh personal computers (STAT VIEW-J 4.11; SAS Institute, Cary, NC). One-way factorial ANOVA, followed by Sheffe’s multiple-comparison test, was also performed. P <0.05 was considered significant.

RESULTS

TUNEL-positive Cardiomyocytes in the Course of Global Ischemia/Reperfusion

In a TUNEL-stained microscopic field (Fig. 2, A–F), TUNEL-positive cardiomyocyte nuclei were increased from 2 to 8 and 24 hr after reperfusion in the warm I/R group (Fig. 2, A–C), but not in the cold one (Fig. 2, D–F). Figure 2G shows the time course for TUNEL-positive cardiomyocyte nuclei (%). The nuclei significantly increased with time after reperfusion, peaked at 24 hr (47.5±9.19%), and had decreased at 48 hr (30.4±9.50%) in the warm I/R group. In contrast, the TUNEL-positive nuclei in the cold I/R group remained at an extremely low level throughout the 48 hr (0.40±0.04%). No TUNEL-positive cardiomyocyte nuclei were detected in control hearts (without I/R) or in the hearts that had undergone ischemia alone (0 hr). Regarding cells other than cardiomyocytes, the numbers of TUNEL-positive fibroblast-like and inflammatory cells were extremely low (0.55±0.12% and 0.58±0.17%, respectively).

Figure 2
Figure 2:
TUNEL-positive cardiomyocytes and genomic DNA electrophoresis during global warm or cold I/R. (A–F) TUNEL staining of ischemia/reperfused rat hearts (original magnification ×400). TUNEL-positive cardiomyocytes were increased from 2 to 8 and 24 hr after warm I/R (A, B, and C) but not after global cold I/R (D, E, and F). (G) Time course of the percentage of TUNEL-positive cardiomyocytes. Open and solid bars indicate the cold and warm I/R groups, respectively. Mean±SE for 6 rats. *P <0.005 vs. corresponding cold I/R group. †P <0.05 vs. 0, 2, 4, 8, and 48 hr warm I/R groups. (H) Genomic DNA electrophoresis on a 1.8 agarose gel. Cold ischemia followed by 24 hr of reperfusion did not induce visible DNA ladders. However, DNA ladders were clearly detected at 8 and 24 hr after reperfusion in the warm I/R group. M indicates a 100 base pair ladder. Data are representative of three experiments.

DNA Fragmentation in the Course of Global Ischemia/Reperfusion

DNA ladders were detected at 8 and 24 hr after reperfusion in the hearts of the warm I/R group (Fig. 2H), indicating the presence of apoptotic DNA fragmentation. As observed on densitometric analysis, the fragmentation of DNA at 24 hr was more frequent than that at 8 hr (1.4-fold, n=3). In contrast, cold ischemia followed by 24 hr of reperfusion did not induce visible DNA ladders (Fig. 2H).

Effects of a Broad-spectrum Caspase Inhibitor and Volume-Regulatory Cl Channel Inhibitors on TUNEL-positivity, DNA Laddering, and Caspase-3-like Activity

The effects of volume-regulatory Cl channel inhibitors (DIDS and NPPB) and a broad-spectrum caspase inhibitor (Z-Asp-DCB) were examined. The percentage of TUNEL-positive cardiomyocytes in the warm I/R group at 24 hr (51.8±11.2%) was significantly decreased by Z-Asp-DCB to 6.68±3.63%, by DIDS to 0.55±0.26%, and by NPPB to 0.89±0.37% (Fig. 3A). DNA electrophoresis demonstrated DNA ladders in a heart sample from the warm I/R (24 hr) group, which disappeared with the administration of Z-Asp-DCB, DIDS, or NPPB (Fig. 3B). We examined the time course of caspase-3-like activity at five points (control hearts, and 4, 8, 14, and 24 hr after I/R). The activity was significantly higher only at 4 hr after reperfusion compared with control hearts. Figure 3C shows that the blockers, Z-Asp-DCB, DIDS, and NPPB, decreased the caspase-3-like activity in the 4-hr reperfused hearts.

Figure 3
Figure 3:
Effects of a broad-spectrum caspase inhibitor and volume-regulatory Cl− channel inhibitors on global ischemia followed by 24 hr of reperfusion. (A) Percentages of TUNEL-positive myocytes in transplanted hearts administered the inhibitors. Treatment with a broad-spectrum caspase inhibitor (+ Z-Asp-DCB [24 hr] group, n=6) or a volume-regulatory Cl− channel inhibitor (+ DIDS [24 hr] or + NPPB [24 hr] group, n=6 each) resulted in a decrease in the TUNEL-positive cardiomyocytes compared with in the vehicle-treated warm I/R (24 hr) group (*P <0.001, n=7). (B) Administration of the inhibitors prevented DNA ladders. (C) Global warm I/R increased caspase-3-like activity in the transplanted hearts at 4 hr after reperfusion (warm I/R [4 hr]) approximately 2.5-fold compared with in control hearts without I/R (*P <0.001, n=3). The inhibitors significantly decreased the caspase-3-like activity in the 4-hr reperfused hearts (†P <0.005, n=3 each).

Functional Assessment of Global Ischemia/ Reperfused Hearts

To assess the effects of the inhibitors on cardiac function, we measured FS of transplanted hearts by ultrasound cardiography. As shown in Figure 4, warm I/R resulted in severe impairment in the FS at 24 hr after reperfusion (6.07±1.22%, n=7) compared with that in the case of cold I/R (22.3±2.70%, n=6). The administration of Z-Asp-DCB, DIDS, or NPPB improved the FS (18.2±1.58%, 21.4±1.68%, or 21.5±1.93%, respectively, n=6 each).

Figure 4
Figure 4:
Effects of a broad-spectrum caspase inhibitor and volume-regulatory Cl− channel inhibitors on FS of the left ventricle at 24 hr after reperfusion. Global warm I/R markedly decreased the FS after 24 hr of reperfusion (warm I/R [24 hr]), which was improved by these inhibitors to nearly the level with global cold ischemia followed by 24 hr of reperfusion (cold I/R [24 hr]). The FS was measured by ultrasound cardiography through the abdominal walls of the recipient rats. Mean±SE for 6 rats. *P <0.005 vs. warm I/R (24 hr).

Effects of a Broad-spectrum Caspase Inhibitor and Volume-Regulatory Cl Channel Inhibitors on the TTC-Unstained Area

Figure 5, A–D are representative views of TTC-stained heart samples in the warm I/R (24 hr), + Z-Asp-DCB (24 hr), + DIDS (24 hr), and + NPPB (24 hr) groups, respectively. Figure 5E shows the TTC-unstained areas of transversely sectioned left ventricles as percentages. The TTC-unstained area in the warm I/R (24 hr) group (58.8±11.4%) decreased with the administration of Z-Asp-DCB, DIDS, or NPPB (14.8±8.27%, 3.89±1.00%, or 4.20±0.90%, respectively). TTC staining has been used to determine the infarcted area, i.e., necrotic cells. However, TTC-unstained areas have been reported to contain areas of reversibly-injured or apoptotic myocytes as well as necrotic myocytes, as found with the combination of HRP infiltration assaying, TTC staining, and TUNEL staining, where injected HRP passed through the ruptured membranes of necrotic cells and slightly permeated apoptotic cells (18,19). We examined our tissue samples at 24 hr after reperfusion with this method. According to the above reports (18,19), viable myocytes should be TTC-positive, and HRP- and TUNEL-negative. Apoptotic myocytes should be TTC-negative, slightly HRP-positive (faint green cytoplasm), and TUNEL-positive (brown nuclei). Necrotic myocytes should be TTC-negative, strongly HRP-positive (deep green cytoplasm), and TUNEL-negative. Reversibly injured myocytes should be TTC-, HRP-, and TUNEL-negative. In a frozen section (magnification ×400), TTC-positive myocytes showed regular snowy-crystalline bodies in the cytoplasm (Fig. 5F) and were neither HRP- nor TUNEL-negative. The cytoplasm of cells in TTC-unstained areas was stained with HRP slightly (faint green, Fig. 5G) or strongly (deep green, Fig. 5H). Most of the slightly HRP-positive myocytes were TUNEL-positive, whereas strongly HRP-positive myocytes were TUNEL-negative. In TTC-unstained areas, both HRP- and TUNEL-negative myocytes were scarce.

Figure 5
Figure 5:
Effects of a broad-spectrum caspase inhibitor and volume-regulatory Cl− channel inhibitors on cell viability and membrane integrity. (A) Global warm I/R stress-induced TTC-unstained areas in the 24-hr-reperfused hearts (warm I/R [24 hr]). Z-Asp-DCB (B), DIDS (C), or NPPB (D) significantly decreased the TTC-unstained areas (original magnification ×1). (E) The percentage of the TTC-unstained area versus the total left ventricle area. The inhibitors significantly reduced the TTC-unstained area. *P <0.005 vs. warm I/R (24 hr) group. (F–H) HRP infiltration assaying, TTC staining, and TUNEL staining were performed simultaneously on the same frozen section (10-μm thick; original magnification ×400). HRP was administered intravenously to recipient rats to assess the membrane rupture or permeability of myocytes. (F) TTC-positive areas, which are HRP- and TUNEL-negative. Reddish snowy-crystalline bodies can be seen in the TTC-stained cytoplasm (arrows). (G) TUNEL-positive myocytes (brown-colored nuclei, arrowheads), which are slightly HRP-positive and TTC-negative. The HRP-positive signal is green in color in the cytoplasm of myocytes. (H) Strongly HRP-positive myocytes, which are TUNEL- and TTC-negative.

DISCUSSION

The current study demonstrated that reperfusion (via transplantation to the abdominal aortae of recipient rats) after global 8-min warm ischemia of the heart induced caspase activation, TUNEL-positive cardiomyocyte nuclei, and DNA ladders in vivo. A novel finding in this study is that blockers of volume-regulatory Cl channels, NPPB and DIDS, markedly decreased caspase activation, TUNEL-positive cardiomyocyte nuclei, DNA ladders, and TTC-unstained areas and improved the FS of the left ventricle after global I/R, similar to a caspase inhibitor. On the contrary, after cold ischemia, reperfusion of the heart did not induce the appearance of TUNEL-positive cardiomyocyte nuclei, TTC-unstained areas, or DNA ladders.

DNA Fragmentation Induced by Global Warm Ischemia/Reperfusion

The lack of an increase in TUNEL-positive nuclei in the cold ischemia group suggests that the preservation of transplanted hearts at body temperature even for 8 min induces myocardial DNA fragmentation and functional failure. Whereas hypothermia decreases aerobic mitochondrial metabolism (20), a higher temperature increases mitochondrial metabolism and, during ischemia, yields fully reduced mitochondrial cytochrome (21) and intermediate electron transport components, such as ubisemiquinone (22). Reperfusion may increase superoxide generation through the reaction between ubisemiquinone and oxygen (22), result in the loss of mitochondrial barrier function, and trigger a part of the apoptotic mechanism.

In our preliminary study, a 15-min warm ischemic period followed by reperfusion produced severe infarction with no TUNEL-positivity and no hematoxylin staining of nuclei in the heart, whereas a 5-min warm ischemic period followed by reperfusion also induced no TUNEL-positivity in the heart (well stained with TTC). The present transplanted hearts had a low pressure and volume load in the left ventricle because of the Langendorff mode. In the present model, anti-apoptotic intervention reduced caspase activation, TUNEL-positive cardiomyocytes, and TTC-unstained areas. The 8-min warm ischemia followed by reperfusion in the present model would be a moderate stimulus, like in the case of the peri-infarct zone of the severe regional ischemia model (4,5), because the occurrence of apoptosis is more typical of moderate ischemia with low levels of adenosine triphosphate (ATP), whereas oncosis is typical of total ATP depletion after severe ischemia.

TTC Staining, TUNEL Staining, and DNA Electrophoresis

TTC staining has been demonstrated to be well correlated with the results of histological assessment of infarct size, in both a model of severe regional ischemia due to coronary occlusion (23) and one of global ischemia (17), and has been widely used to verify the cardioprotective effects of various interventions. Because a normal myocardium is stained red with TTC (24,25), loss of TTC-positivity seems to be an excellent marker for visualizing an ischemic change, as markedly shown in the present study, which is not necessarily due to only necrotic cells. The myocytes in reversible injury, necrosis, and apoptosis are not stained with TTC (18,19), because loss of TTC-positivity is basically thought to be an index of severe mitochondrial damage and/or loss of substrates from leaky mitochondria and leaky cells, there consequently being no substrate for oxidation of the tetrazolium dye.

The heterogeneous mixture of cells in reversible injury will include cells at the late irreversible apoptotic stage to the oncotic necrotic stage after 24-hr reperfusion and vice versa, i.e., TUNEL-positive myocytes could exhibit a transient apoptotic stage before entering or during the necrotic stage in the acute infarcted heart. Pretreatment with reagents that prevent the early reversible apoptotic process is therefore assumed to have protected the reperfused hearts from damage in the present study. The specificity of the TUNEL assay for measurement of apoptosis has been debated for the hearts that have undergone I/R (8). However, the TUNEL method must be considered a sensitive and convenient one for detecting apoptotic cells so long as alternative techniques are also applied (26). The presence of cells undergoing from apoptosis to oncotic necrosis, especially on I/R of the heart, must be related to that the TUNEL method has been reported to label necrotic myocytes (27).

The detection of DNA laddering on agarose gel electrophoresis is extremely sensitive, such that double-stranded oligonucleosomal fragments can be detected when as few as 2% apoptotic cells are observed morphologically among cultured cells (28). DNA has been extracted from heart tissue, including myocytes and nonmyocytes such as coronary endothelial cells, interstitial macrophages, fibroblasts, and neutrophils. In the present study, however, because most of the TUNEL-positive cells originated from myocytes (the percentage of TUNEL-positive noncardiomyocytes vs. total cell number was 0.58±0.34%), the ladder pattern should mainly reflect DNA fragmentation of cardiomyocytes.

Simultaneous HRP Infiltration Assaying, TTC Staining, and TUNEL Staining

At an early time after a lower degree of I/R, a significant part of heart tissue may be composed of reversibly injured cells, which will have become irreversibly injured cells after 24-hr reperfusion. Because the current study demonstrated that some inhibitors ameliorated the cardiac function, we speculate that they blocked the conversion of early reversibly injured cells into late irreversibly injured cells, which allowed cells to recover during 24 hr after the damage and thus led to a decrease in the TTC-unstained area. We then performed HRP infiltration assaying, TTC staining, and TUNEL staining simultaneously to characterize the viable, apoptotic, necrotic, or reversibly injured myocytes in the TTC-unstained areas at 24-hr reperfusion. For the HRP infiltration assay, HRP was injected to examine cell membrane permeability or rupture. Among TTC-unstained cells, TUNEL-positive myocytes showed a weak signal from infiltrated HRP, suggesting altered membrane permeability of apoptotic myocytes (18,19). Although TTC-unstained TUNEL-negative myocytes showed a strong signal from HRP that passed through the ruptured membrane (necrotic myocytes), TTC-stained myocytes, i.e., viable myocytes, showed no HRP presence. Administration of apoptosis inhibitors suppressed activation of caspase-3 at 4 hr and reduced the number of TUNEL-positive myocytes from approximately 50% to 1–6%. These results suggest that the recovered fraction of TUNEL-positive myocytes comprised apoptotic myocytes, at least at the early stage, assuming that these inhibitors did not affect necrotic myocytes.

Volume-regulatory Cl Channel Inhibitors and Apoptosis

The present study suggests that volume-regulatory Cl channels are one of the drivers of apoptotic cardiomyocyte death in vivo, because volume-regulatory Cl channel inhibitors, DIDS and NPPB, were effective in reducing caspase-3 activation, DNA laddering, TUNEL-positive cardiomyocytes, and TTC-unstained areas and in restoring the impaired FS of the left ventricle induced by global I/R. Apoptosis occurs with normotonic shrinkage of cells. Volume-regulatory Cl or K+ channels are activated by cell swelling in a large variety of cell types. Induction of an apoptotic volume decrease (AVD) under normotonic conditions was found to be coupled with facilitation of the regulatory volume decrease (RVD), which is known to be attained through parallel operation of Cl and K+ channels under hypotonic conditions (12). Both the AVD induction and RVD facilitation were found to precede cytochrome c release, caspase-3 activation, DNA laddering, and ultrastructural alterations in cultured cell lines after stimulation with staurosporine or tumor necrosis factor plus cycloheximide. When volume-regulatory Cl channels were blocked, the cells did not show subsequent apoptotic biochemical and morphological events and thus were protected from death. Although the present results could not show the AVD due to the in vivo conditions, the volume-regulatory Cl channel inhibitors must have been similarly effective in preventing the AVD, which is the prerequisite for apoptosis or the apoptotic process leading to the death of cardiomyocytes. Recently, Vu et al. (29) demonstrated the involvement of caspases in regulation of K+ efflux and the coupled apoptotic volume decrease.

Type of Cell Death in Ischemia/Reperfused Heart

Ohno et al. (27) recently found, using electron microscopic TUNEL staining, that TUNEL-positive myocytes are actually cells undergoing oncosis because of colocalization of plasma membrane rupture. Studies (3,30,31), however, have revealed the typical ultrastructural morphology of apoptotic changes in the reperfused myocardium on TUNEL staining and electron microscopy. Furthermore, activation of the typical apoptotic process has been demonstrated in the reperfused heart in vivo (6,14,32). Because apoptosis requires energy, because of the depletion of ATP in severely ischemic hearts, the apoptotic program cannot be completed, which eventually leads to necrosis. Some steps of the apoptotic program might be activated during I/R-induced oncotic cell death (33,34). The ratio of the apoptotic and oncotic processes in the cardiomyocyte death process must depend on both the initial ischemic injury and variable stress during reperfusion, i.e., left ventricular load, systemic conditions, and neutrophil activity.

Formigli et al. (35) recently showed that the ultrastructural shape on cell death changed from that of apoptosis to that of necrosis with increasing intensity of the hypoxic insult. More than true necrosis, an intermediate form of cell death between apoptosis and necrosis, namely “aponecrosis,” was predominant in the cultured cells exposed to high levels of a hypoxic stimulus. Furthermore, in most of the dying cells, molecular and morphological signs of apoptosis (e.g., oligonucleosomal DNA fragmentation and nuclear degradation into small electron-dense chromatin bodies) coexisted with degenerative necrotic features including cytoplasmic swelling and plasma membrane disruption. So, they concluded that the classical apoptosis and necrosis might represent only two extremes of a continuum of intermediate forms of cell death. Similarly, reperfused myocytes in a beating heart cannot maintain the intracellular ATP level to continue the apoptotic process, which leads to rupture of the plasma membrane. This may mean an “aponecrosis” situation in reperfused myocytes. The rate of decrease in ATP may play an important role in determining whether myocytes undergo apoptosis or necrosis in response to I/R or other stimuli.

Implication for Clinical Transplantation

Cold I/R induced little cardiomyocyte apoptosis in the present model. For heart transplantation, donor hearts are maintained cold until reperfusion; however, the temperature of the donor heart may not always be ∼4°C throughout the transplantation technique if the donor heart is positioned in the mediastinum of the recipient. The clinically acceptable ischemic period for heart transplantation still remains at 4–8 hr. It may be useful to block volume-regulatory chloride channels in the donor heart if the period until reperfusion is longer than 4–8 hr.

Chloride channel inhibitors reduce the norepinephrine-induced increase in mean arterial pressure for a short period in rats (36). It is, therefore, necessary to inject the blockers into the donor and recipient slowly, because rapid intravenous injection may result in hypotension. It has been reported that the onset of inhibition was very fast (within 1 sec after administration) (37,38) and that the effect remained at even 3–6 hr after administration (36,39). The blockers were injected twice, i.e., into the donor and the recipient in the present study; however, it is possible that the effect may be attained with lower concentrations of the inhibitors on continuous intravenous injection.

The present results demonstrated that global I/R of the heart induces cardiomyocyte DNA fragmentation when the temperature during the ischemic stress is elevated. The present work has increased our understanding of the apoptosis in I/R of the heart by demonstrating that blockers of volume-regulatory Cl channels are as able as caspase inhibitors to improve the cardiac function and to reduce the DNA fragmentation, TTC-unstained area, and caspase-3-like activity.

REFERENCES

1. Gottlieb RA, Burleson KO, Kloner RA, Babior BM, Engler RL. Reperfusion injury induces apoptosis in rabbit cardiomyocytes. J Clin Invest 1994; 94: 1621.
2. Kajstura J, Cheng W, Reiss K, et al. Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 1996; 74: 86.
3. Umansky SR, Tomei LD. Apoptosis in the heart. Adv Pharmacol 1997; 41: 383.
4. Saraste A, Pulkki K, Kallajoki M, Henriksen K, Parvinen M, Voipio Pulkki LM. Apoptosis in human acute myocardial infarction. Circulation 1997; 95: 320.
5. Yaoita H, Ogawa K, Maehara K, Maruyama Y. Attenuation of ischemia/reperfusion injury in rats by a caspase inhibitor. Circulation 1998; 97: 276.
6. Formigli L, Ibba Manneschi L, Perna AM, et al. Ischemia-reperfusion-induced apoptosis and p53 expression in the course of rat heterotopic heart transplantation. Microvasc Res 1998; 56: 277.
7. Freude B, Masters TN, Robicsek F, et al. Apoptosis is initiated by myocardial ischemia and executed during reperfusion. J Mol Cell Cardiol 2000; 32: 197.
8. Buja LM, Entman ML. Modes of myocardial cell injury and cell death in ischemic heart disease. Circulation 1998; 98: 1355.
9. Yaoita H, Ogawa K, Maehara K, Maruyama Y. Apoptosis in relevant clinical situations: contribution of apoptosis in myocardial infarction. Cardiovasc Res 2000; 45: 630.
10. Gottlieb RA, Engler RL. Apoptosis in myocardial ischemia-reperfusion. Ann N Y Acad Sci 1999; 874: 412.
11. Rasola A, Farahi Far D, Hofman P, Rossi B. Lack of internucleosomal DNA fragmentation is related to Cl efflux impairment in hematopoietic cell apoptosis. FASEB J 1999; 13: 1711.
12. Maeno E, Ishizaki Y, Kanaseki T, Hazama A, Okada Y. Normotonic cell shrinkage because of disordered volume regulation is an early prerequisite to apoptosis. Proc Natl Acad Sci U S A 2000; 97: 9487.
13. Okamura T, Miura T, Takemura G, et al. Effect of caspase inhibitors on myocardial infarct size and myocyte DNA fragmentation in the ischemia-reperfused rat heart. Cardiovasc Res 2000; 45: 642.
14. Holly TA, Drincic A, Byun Y, et al. Caspase inhibition reduces myocyte cell death induced by myocardial ischemia and reperfusion in vivo. J Mol Cell Cardiol 1999; 31: 1709.
15. Ono K, Lindsey ES. Improved technique of heart transplantation in rats. J Thorac Cardiovasc Surg 1969; 57: 225.
16. Kasahara Y, Tuder RM, Taraseviciene-Stewart L, et al. Inhibition of VEGF receptors causes lung cell apoptosis and emphysema. J Clin Invest 2000; 106: 1311.
17. Sumeray MS, Yellon DM. Characterisation and validation of a murine model of global ischaemia-reperfusion injury. Mol Cell Biochem 1998; 186: 61.
18. Takashi E, Wang Y, Ashraf M. Activation of mitochondrial K(ATP) channel elicits late preconditioning against myocardial infarction via protein kinase C signaling pathway. Circ Res 1999; 85: 1146.
19. Takashi E, Ashraf M. Pathologic assessment of myocardial cell necrosis and apoptosis after ischemia and reperfusion with molecular and morphological markers. J Mol Cell Cardiol 2000; 32: 209.
20. Seiyama A, Kosaka H, Maeda N, Shiga T. Effect of hypothermia on skeletal muscle metabolism in perfused rat hindlimb. Cryobiology 1996; 33: 338.
21. Chen SS, Yoshihara H, Harada N, et al. Measurement of redox states of mitochondrial cytochrome aa3 in regions of liver lobule by reflectance microspectroscopy. Am J Physiol 1993; 264: G375.
22. Chandel NS, Maltepe E, Goldwasser E, Mathieu CE, Simon MC, Schumacker PT. Mitochondrial reactive oxygen species trigger hypoxia-induced transcription. Proc Natl Acad Sci U S A 1998; 95: 11715.
23. Fishbein MC, Meerbaum S, Rit J, et al. Early phase acute myocardial infarct size quantification: validation of the triphenyl tetrazolium chloride tissue enzyme staining technique. Am Heart J 1981; 101: 593.
24. Vivaldi MT, Kloner RA, Schoen FJ. Triphenyltetrazolium staining of irreversible ischemic injury following coronary artery occlusion in rats. Am J Pathol 1985; 121: 522.
25. Klein HH, Puschmann S, Schaper J, Schaper W. The mechanism of the tetrazolium reaction in identifying experimental myocardial infarction. Virchows Arch [A] 1981; 393: 287.
26. Mesner PW Jr, Kaufmann SH. Methods utilized in the study of apoptosis. Adv Pharmacol 1997; 41: 57.
27. Ohno M, Takemura G, Ohno A, et al. “Apoptotic” myocytes in infarct area in rabbit hearts may be oncotic myocytes with DNA fragmentation: analysis by immunogold electron microscopy combined with in situ nick end-labeling. Circulation 1998; 98: 1422.
28. Collins RJ, Harmon BV, Gobe GC, Kerr JF. Internucleosomal DNA cleavage should not be the sole criterion for identifying apoptosis. Int J Radiat Biol 1992; 61: 451.
29. Vu CC, Bortner CD, Cidlowski JA. Differential involvement of initiator caspases in apoptotic volume decrease and potassium efflux during Fas- and UV-induced cell death. J Biol Chem 2001; 276: 37602.
30. Freude B, Masters TN, Kostin S, Robicsek F, Schaper J. Cardiomyocyte apoptosis in acute and chronic conditions. Basic Res Cardiol 1998; 93: 85.
31. Sharov VG, Sabbah HN, Shimoyama H, Goussev AV, Lesch M, Goldstein S. Evidence of cardiocyte apoptosis in myocardium of dogs with chronic heart failure. Am J Pathol 1996; 148: 141.
32. Black SC, Huang JQ, Rezaiefar P, et al. Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat. J Mol Cell Cardiol 1998; 30: 733.
33. Shimizu S, Eguchi Y, Kamiike W, Matsuda H, Tsujimoto Y. Bcl-2 expression prevents activation of the ICE protease cascade. Oncogene 1996; 12: 2251.
34. Shimizu S, Eguchi Y, Kamiike W, et al. Bcl-2 blocks loss of mitochondrial membrane potential while ICE inhibitors act at a different step during inhibition of death induced by respiratory chain inhibitors. Oncogene 1996; 13: 21.
35. Formigli L, Papucci L, Tani A, et al. Aponecrosis: morphological and biochemical exploration of a syncretic process of cell death sharing apoptosis and necrosis. J Cell Physiol 2000; 182: 41.
36. Lamb FS, Kooy NW, Lewis SJ. Role of Cl(-) channels in alpha-adrenoceptor-mediated vasoconstriction in the anesthetized rat. Eur J Pharmacol 2000; 401: 403.
37. Tilmann M, Kunzelmann K, Frobe U, et al. Different types of blockers of the intermediate-conductance outwardly rectifying chloride channel in epithelia. Pflugers Arch 1991; 418: 556.
38. Okada Y. Volume expansion-sensing outward-rectifier Cl- channel: fresh start to the molecular identity and volume sensor. Am J Physiol 1997; 273: C755.
39. Horie S, Yano S, Watanabe K. Inhibition of gastric acid secretion in vivo and in vitro by an inhibitor of Cl(-)-HCO3- exchanger, 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid. J Pharmacol Exp Ther 1993; 265: 1313.
© 2002 Lippincott Williams & Wilkins, Inc.