The release of cytochrome c from the mitochondria to the cytosol is a critical step for downstream caspase-mediated apoptotic signal transduction in ischemia-reperfusion (I/R)-induced myocardial tissue injury. 10-N-nonyl acridine orange (NAO), a cardiolipin-specific dye, has been shown to inhibit Bid-mediated cytochrome c release from isolated mitochondria in vitro; however, the possible protective effects of NAO and the mechanisms underlying the protection from myocardial I/R-induced tissue injury in a rat model are unknown. Male Sprague-Dawley rats were subjected to a 30-min coronary arterial occlusion followed by reperfusion. All rats received either vehicle or NAO (100 μg/kg iv) 10 min before the occlusion. The infarct size in the heart at 24 h after reperfusion was significantly reduced in NAO-treated rats compared with vehicle-treated rats. NAO treatment significantly reduced the cytosolic cytochrome c contents and caspase-9 activity in the ischemic region but did not affect caspase-8 activity. Furthermore, NAO treatment markedly suppressed the translocation of truncated Bid, a proapoptotic Bcl-2 family member, to the mitochondrial fraction. NAO also suppressed the mitochondrial swelling and oxygen uptake stimulated by calcium overload. The results suggest that NAO possesses protective effects against myocardial I/R injury, which may be due to the suppression of cytochrome c release through blockade of truncated Bid translocation to mitochondria and inhibition of the opening of mitochondrial permeability transition pores.
apoptosis, a special form of cell death that differs from necrosis in many aspects, participates in the cardiomyocyte death observed in ischemia-reperfusion (I/R)-induced tissue injury (8, 12, 18, 31). The most widely recognized biochemical features of apoptosis during I/R procedures are the release of cytochrome c from the mitochondria and the activation of a class of cysteine proteases called caspases, including caspase-3, caspase-8, and caspase-9 (8, 33, 37). Two mechanisms have been considered to mediate I/R-induced apoptosis in tissues or cells. First, opening of mitochondrial permeability transition pores (mPTPs), which is activated by a variety of stimuli including hypoxia and oxidative stress, promotes cytochrome c release from the intermembrane spaces of mitochondria into the cytosol, leading to the formation of the apoptosome complex and the subsequent activation of caspase-9 (2, 4, 14). In fact, inhibition of mPTP opening by cyclosporine A has been shown to be cardioprotective against I/R injury by preventing the increase in cytosolic cytochrome c and subsequent augmentation of caspase activities (12–13, 28).
Second, although the contribution of the death ligand/death receptor pathway in I/R-induced tissue injury is still controversial (17, 21, 36), several reports have described that caspase-8, an isoenzyme responsible for death receptor signal transduction, is activated after I/R stimuli (34). Activated caspase-8 also participates in cytochrome c release from the mitochondria through a pathway mediated by Bid, a proapoptotic member of the Bcl-2 family, and, notably, this pathway is independent of mPTP opening. Recently, it has been demonstrated that, after cleavage by caspase-8, a truncated form of Bid (tBid) cleaved by caspase-8 is translocated to the mitochondria, where it binds to mitochondrial membranes and interacts directly with cardiolipin, a mitochondria-directed phospholipid, and mediates mitochondrial damage in the Fas pathway of apoptosis (11, 22, 24, 26). However, the precise role and contribution of tBid in I/R procedures are poorly understood.
A cardiolipin-specific dye, 10-N-nonyl acridine orange (NAO), can preferentially suppress tBid binding to the mitochondria and inhibit Bid-induced cytochrome c release from isolated mitochondria (9, 19, 27). Cardiolipin is an essential phospholipid for maintaining the mitochondrial structure and the functions of various mitochondrial proteins (15, 32, 35, 39). However, few studies have investigated NAO actions using living cells or isolated mitochondria, and none of these studies investigated the possible effects of NAO in a whole body study. In the present study, we examined the possible cardioprotective effects of NAO against myocardial I/R-induced tissue injury using a rat in vivo model by measuring cytosolic cytochrome c levels and caspase activities (caspase-8 and caspase-9) in hearts subjected to an I/R procedure. To further explore the mechanisms responsible for the actions of NAO, we investigated Bid cleavage and its translocation to the mitochondrial fraction as well as the involvement of mPTP opening.
MATERIALS AND METHODS
Animal preparation and treatment.
Nine-week-old male Sprague-Dawley rats (Clea) were used in this study. The cardiac I/R model has been previously described (20). Briefly, under pentobarbital anesthesia (50 mg/kg ip), the heart was exposed via a left thoracotomy performed at the fifth intercostal space followed by passing of a ligature (6-0 proline) below the left descending coronary artery. Regional left ventricular ischemia was performed for 30 min via occlusion of the coronary artery by clamping it together with the propylene tube. At 10 min before the ischemia, NAO (100 μg/kg) or vehicle (5% DMSO) was randomly given intravenously over 2 min. We confirmed that this dose had minimal effects on the mean arterial blood pressure and heart rate. Reperfusion was confirmed by visualizing an epicardial hyperemic response. Hearts were excised under anesthesia at the indicated time points and perfused with ice-cold PBS. Twenty-four h after reperfusion, excised hearts were perfused with fluorescent microspheres (Duke Scientific) from coronary artery under occlusion of the left descending coronary artery with the remaining sutures. Hearts were traversely cut along the ligation point. The ischemic regions (area at risk) were recorded under ultraviolet light. Ratios of the ischemic area to the total area were calculated. After identification of the ischemic region using microspheres, cross sections of the cardiac tissue (1–2 mm thick) were incubated in a 1.25% solution of triphenyl tetrazolium chloride in PBS at 37°C for 10 min, and infarct areas were recorded. The ratios of the infarct area to the total area were calculated. The ratios of the infarct area to the area at risk were obtained. All surgical and experimental procedures were approved by and performed according to guidelines for the care and use of animals established by Kagawa University.
Western blot analyses of cytosolic cytochrome c and tBid in myocardium.
Cytosolic fractions were separated with a previously described method (34). Briefly, immediately after reperfusion, cardiac tissues were gently homogenized with a Dounce homogenizer in cytosolic extraction buffer (250 mM sucrose, 70 mM KCl, 137 mM NaCl, 4.3 mM Na2HPO4, 1.4 mM KH2PO4, 1 mM PMSF, 10 μg/ml aprotinin, 20 μg/ml leupeptin, and 250 mg/ml digitonin). Next, lysates were centrifuged at 600 g for 10 min at 4°C, and the resulting supernatant was centrifuged at 10,000 g for 10 min to yield a cytosolic extract (supernatant) and mitochondria (pellet). The mitochondrial pellet was washed three times and resuspended in lysis buffer [20 mM Tris·HCl (pH 7.4), 140 mM NaCl, 1% Triton-X, 10% glycerol, 1 mM β-glycerophosphate, and 1 mM sodium orthovanadate]. Aliquots of the supernatant containing 20 μg protein were supplemented with 5× SDS-PAGE loading buffer, subjected to 12% SDS-PAGE, and transferred to nitrocellulose membranes. Membranes were probed with an anti-Bid or anti-cytochrome c antibody followed by a horseradish peroxidase-conjugated secondary antibody and subjected to autoradiography with enhanced chemiluminescence. Immunoblots were normalized by β-actin for the cytosolic fraction and prohibitin for the mitochondrial fraction.
Measurement of caspase-8 and caspase-9 activities.
The activities of cardiac caspase-8 and caspase-9 were measured using caspase colorimetric assay kits (Chemicon) following the manufacturer's instructions. Briefly, cardiac tissue was homogenized in ice-cold lysis buffer. After the centrifugation of the homogenates at 10,000 g for 5 min at 4°C, the supernatants were collected, and their protein concentrations were measured with protein assay kits (Bio-Rad). Aliquots of the supernatants containing 200 μg protein were loaded into individual wells of a 96-well plate and incubated with 25 μg of Ac-IETC-pNA or Ac-LEHD-pNA at 37°C for 1.5 h. The pNA moiety was cleaved from IETC (by caspase-8) or LEHD (by caspase-9), and the free pNA was quantified using a microplate spectrophotometer at 405 nm.
Mitochondrial swelling and oxygen uptake.
Mitochondria isolated from the cardiac left ventricle were prepared essentially as previously described (38). The protein concentration of the mitochondrial solution was adjusted to 10 μg/μl as measured by the protein assay kits (Bio-Rad). Mitochondrial swelling was qualitatively determined by measuring the apparent absorbance decreases of mitochondrial suspensions at 540 nm in a model U-2800 spectrophotometer (Hitachi). The oxygen consumption was measured polarographically at 25°C with a Clark-type oxygen mini-electrode (Hansatech) using an air-saturated incubation buffer [250 mM sucrose, 5 mM succinate, 5 mM Pi, 1.25 μM rotenone, and 5 mM MOPS (pH 7.4)] and a water-jacketed reaction chamber (1.5 ml) (20).
All data are presented as means ± SE. Statistical significance between more than two groups was tested using two-way ANOVA followed by the Newman-Keuls test or an unpaired two-tailed Student's t-test as appropriate. P values of <0.05 were considered statistically significant.
Sources of materials.
NAO was purchased from Invitrogen. Antibodies were purchased from Santa Cruz Biotechnology. All other chemicals were of reagent grade, purchased from commercial sources, and used without further purification.
Effect of NAO on infarct size after the cardiac I/R procedure.
The ratios of infarct area to area at risk in I/R hearts are shown in Fig. 1. In vehicle-treated rats, occlusion of the left descending coronary artery for 30 min resulted in an infarct size of 37 ± 2% of the area at risk determined at 24 h after reperfusion. Pretreatment with NAO significantly reduced the infarct area-to-area at risk ratio to 10 ± 3%, indicating that pretreatment with NAO confers protective effects on myocardial I/R-induced tissue injury.
Time course of cytochrome c release during I/R.
Before exploring the mechanisms responsible for the protective effects of NAO, we first determined the time course of changes of cytochrome c release into the cytosol and Bid cleavage during the I/R procedure by Western blot analysis. As shown in Fig. 2, the cytochrome c contents in the cytosolic fraction of the ischemic region increased significantly after reperfusion, with a peak induction of 3.8-fold compared with the level in sham-operated control rats at 3 h. In parallel with the increase of cytochrome c contents in the cytosolic fraction, decreases of cytochrome c in the mitochondrial fraction after reperfusion were observed. Furthermore, 30 min of ischemia alone did not affect the cytochrome c contents of the cytosolic fraction, consistent with a previous report (34).
Time course of Bid truncation during I/R.
The time course of changes in the contents of Bid and tBid in the cytosolic fraction of the ischemic region are shown in Fig. 3. Similar to the results for cytochrome c release, the tBid contents in the cytosolic fraction increased significantly after reperfusion, with a maximal induction of 4.2-fold compared with sham-operated control rats at 6 h. Interestingly, the Bid contents in the cytosolic fraction decreased during the early reperfusion phase (until 3 h after reperfusion), returned to the basal level at 6 h, and then significantly increased at 24 h after reperfusion. According to the results of these time course experiments, the effects of NAO were analyzed using cardiac tissue obtained at 3 h after reperfusion.
Effects of NAO on cytochrome c release and caspase activities.
As shown in Fig. 4A, the increased cytochrome c contents in the cytosolic fraction of the ischemic region at 3 h after reperfusion was significantly suppressed in NAO-treated rats compared with vehicle-treated rats (2.0 ± 0.2- vs. 3.6 ± 0.3-fold). Meanwhile, the decrease of cytochrome c in the mitochondrial fraction after 3 h reperfusion was reversed by NAO treatment (0.75 ± 0.2- vs. 0.35 ± 0.1-fold; Fig. 4B). Similar to the increased cytochrome c contents in the cytosolic fraction, caspase-9 activity in the ischemic region at 3 h after reperfusion was 2.7-fold higher than that in sham-operated control rats. The increased caspase-9 activity was significantly suppressed by pretreatment with NAO to 1.7-fold of sham-operated control rats (Fig. 5A). Caspase-8 activity in the ischemic region at 3 h after reperfusion was increased by approximately twofold compared with sham-operated control rats, but, unlike the case for caspase-9, the increased activity of caspase-8 was not affected by NAO (Fig. 5B). It is of note that neither caspase-9 nor caspase-8 in the nonischemic region was influenced by NAO.
Effects of NAO on tBid translocation to mitochondria.
Reflecting the caspase-8 activity, pretreatment with NAO did not affect the tBid contents in the cytosolic fractions of the ischemic region at 3 h after reperfusion (Fig. 6A). The amount of tBid in the mitochondrial fraction of the ischemic region at 3 h after reperfusion was 4.3-fold higher than that in sham-operated control rats. Furthermore, NAO pretreatment markedly reduced this increase to 1.2 ± 0.2-fold of sham-operated control rats (Fig. 6B), indicating that NAO could prevent tBid translocation to the mitochondria without affecting Bid cleavage.
Effects of NAO on calcium-induced mitochondrial swelling and oxygen uptake.
To test whether NAO modulates the opening of mPTPs, we examined calcium-induced swelling and oxygen uptake using isolated cardiac mitochondria. As shown in Fig. 7, the addition of 50 μM Ca2+ to suspended mitochondria decreased the absorbance at 540 nm, and this effect was apparently inhibited by NAO pretreatment, indicating that NAO was able to suppress the mitochondrial swelling induced by calcium overload. The inhibitory effect of NAO was dose dependent, with an IC50 value of 3.4 μM. Furthermore, as shown in Fig. 8, the calcium-induced increase in mitochondrial oxygen uptake was also suppressed by NAO pretreatment with similar concentrations to those inhibiting mitochondrial swelling.
Recent studies have demonstrated an important contribution of mitochondria to the initiation and execution of apoptosis in response to I/R procedures (5, 23, 25, 34). In particular, the release of cytochrome c from the intermembrane spaces of mitochondria is a critical and imperative event for conduction of the downstream apoptotic pathway. Focusing on the Bid dynamics, we assessed the effect of NAO, a cardiolipin-specific dye, on I/R-induced tissue injury in the rat heart. The results of the present study provide four new important findings. First, pretreatment with NAO can suppress the increases in caspase-9 activity and cytochrome c contents of the cytosolic fraction observed after reperfusion. Second, NAO markedly suppresses I/R-induced tBid translocation to the mitochondria without affecting the increases in caspase-8 activity and total tBid levels. Third, NAO suppresses the mPTP opening induced by calcium overload. Finally, NAO can reduce the infarct size in rat hearts subjected to an I/R procedure.
NAO has a high affinity for cardiolipin, and each cardiolipin molecule can bind to two NAO molecules (30). Although NAO can also bind to other monoacidic phospholipids, such as phosphatidylinositol and phosphatidylserine, at an equal molar ratio, its affinities for these phospholipids are much lower than that for cardiolipin. Therefore, NAO is used for the localization and quantification of cardiolipin in living cells on the assumption of its high specificity (9). Cardiolipin, presented in the inner membrane of mitochondria, is essential for the normal functions of mitochondria because it affects the structural conformations of respiratory complex proteins, adenine nucleotide translocase, carriers, and also cytochrome c (15, 32, 35, 39). Since cardiolipin is composed of highly unsaturated fatty acids, it may be easily peroxidized and may lose its functions under pathophysiological perturbations, such as I/R procedures (29). In fact, peroxidation of cardiolipin in mitochondria results in the dissociation of cytochrome c from the mitochondrial inner membrane and also causes it to lose its binding activity toward NAO (30). The positively charged NAO interacts with cardiolipin in the inner mitochondrial membrane. Although high-dose NAO has been demonstrated to be cytotoxic due to inhibition of inner mitochondrial membrane enzymes, low-dose NAO may block the binding site by other molecules (e.g., tBid) and may also stabilize the mitochondrial membrane structure to prevent the formation of mPTP.
It has been believed that an increase in tBid may lead to increased permeability of the mitochondrial outer membrane through a direct interaction with cardiolipin molecules, thereby resulting in the release of cytochrome c from the intermembrane spaces. In the present study, we demonstrated time-dependent Bid truncation (cleavage) after reperfusion. We found that tBid levels increased time dependently after reperfusion and that the increases in the cytochrome c level in the cytosol followed this trend. Interestingly, the intact Bid level initially decreased and then significantly increased at 24 h after reperfusion, and the involved mechanisms are still under investigation. Kim et al. (9) previously demonstrated that NAO could preferentially suppress tBid binding to mitochondrial contact sites and inhibit Bid-induced mitochondrial cristae reorganization and cytochrome c release using isolated mitochondria. We found that NAO pretreatment could suppress tBid translocation to the mitochondrial fraction in the ischemic region of the rat heart, accompanied by a significant reduction of cytochrome c release, without affecting the Bid activation potency. These results indicate that the Bid pathway may possibly participate, at least in part, in the cytochrome c release and apoptotic process induced by the cardiac I/R procedure used in this study.
Several lines of evidence have accumulated showing that reperfusion may accelerate the opening of mPTPs, another mechanism of cytochrome c release from the mitochondria, thereby dramatically increasing the permeability of the mitochondrial inner membrane to small molecules, including cytochrome c and ions (2, 4, 14). It has been well established that calcium overload or oxidative stress triggers the opening of mPTPs, especially under conditions such as the present cardiac I/R procedure (1, 3, 6, 16), since depletion of nucleotides, increased phosphate levels, and a decrease in the mitochondrial membrane potential accompany calcium overload or oxidative stress. Although the relative contribution of the mPTP to necrosis or apoptosis is still controversial after I/R, our present study demonstrated that NAO, at even a micromolar concentration, can suppress the mitochondrial swelling and augmentation of oxygen consumption induced by calcium overload. NAO also reduced I/R-induced infarct size. Together, these data suggested that NAO might also protect I/R-induced cardiac necrosis by mechanisms of inhibition of mPTP opening and/or tBid translocation.
Consistent with previous reports, the activities of both the initiator caspases, caspase-8 and caspase-9, were increased after reperfusion. Caspase-8 is a well-studied isoenzyme that responds to death receptor apoptotic signaling and may be involved in direct activation of the effector caspase-3 for so-called type I cells or Bid-dependent mitochondrial amplification for so-called type II cells (7). In the present study, the I/R-induced augmentation of caspase-8 activity was not influenced but rather maintained by NAO, whereas the activity of caspase-9 was significantly suppressed by NAO, and this change was in a parallel pattern and consistent with that of the cytochrome c content in the cytosol. Therefore, the predominant downstream pathway from caspase-8 activation induced by I/R procedures may be Bid-dependent cytochrome c release leading to the formation of the apoptosome and the subsequent activation of caspase-9. Further studies are warranted to investigate the potential effects of NAO on other animal models of cardiac remodeling and diseases based on mitochondria-mediated apoptosis.
In summary, we have demonstrated that pretreatment with the cardiolipin-specific dye NAO inhibits the translocation of tBid to the mitochondria induced by I/R stimuli and suppresses the release of cytochrome c from the mitochondria, resulting in the attenuation of the postischemic apoptotic response in the rat heart in vivo. We have also shown that NAO suppresses the mPTP opening and mitochondrial oxygen uptake stimulated by calcium overload. These data indicate that the cardioprotective effects of NAO against postischemic myocardial tissue injury may be based on its inhibitory action toward cytochrome c release by the mitochondria-mediated apoptosis pathway, namely, mPTP opening as well as the Bid-mitochondria association.
This work was supported in part by Ministry of Education, Culture, Sports, Science and Technology of Japan Grant-In-Aid for Scientific Research 19790190. This work was also supported in part by Biotechnology and Biological Sciences Research Council Japan Partnering Program 2007–2011.
No conflicts of interest are declared by the author(s).
- Copyright © 2010 the American Physiological Society