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Am J Physiol Heart Circ Physiol 293: H3348-H3355, 2007. First published October 5, 2007; doi:10.1152/ajpheart.00947.2007
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Acetaminophen-mediated cardioprotection via inhibition of the mitochondrial permeability transition pore-induced apoptotic pathway

Norell M. Hadzimichalis, Sunanda S. Baliga, Roseli Golfetti, Kathryn M. Jaques, Bonnie L. Firestein, and Gary F. Merrill

Department of Cell Biology and Neuroscience, Division of Life Sciences, Rutgers University, Piscataway, New Jersey

Submitted 15 August 2007 ; accepted in final form 5 October 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Our laboratory has previously reported that acetaminophen confers functional cardioprotection following cardiac insult, including ischemia/reperfusion, hypoxia/reoxygenation, and exogenous peroxynitrite administration. In the present study, we further examined the mechanism of acetaminophen-mediated cardioprotection following ischemia/reperfusion injury. Langendorff-perfused guinea pig hearts were exposed to acute treatment with acetaminophen (0.35 mM) or vehicle beginning at 15 min of a 30-min baseline stabilization period. Low-flow global myocardial ischemia was subsequently induced for 30 min followed by 60 min of reperfusion. At the completion of reperfusion, hearts were homogenized and separated into cytosolic and mitochondrial fractions. Mitochondrial swelling and mitochondrial cytochromec release were assessed and found to be significantly and completely reduced in acetaminophen- vs. vehicle-treated hearts following reperfusion. In a separate group of hearts, ventricular myocytes were isolated and subjected to fluorescence-activated cell sorting. Acetaminophen-treated hearts showed a significant decrease in late stage apoptotic myocytes compared with vehicle-treated hearts following injury (58 ± 1 vs. 81 ± 5%, respectively). These data, together with electron micrograph analysis, suggest that acetaminophen mediates cardioprotection, in part, via inhibition of the mitochondrial permeability transition pore and subsequent apoptotic pathway.

mitochondrial swelling; apoptosis; cytochrome c; myocardial ischemia/reperfusion

CONSIDERABLE ATTENTION has been given to the potential detrimental effects of Cox-2-specific analgesics in the mammalian cardiovascular system. However, there have been few rigorous physiological investigations on the effects of other analgesics on the cardiovascular system of mammals, including humans. Acetaminophen (paracetamol, APAP), a popular nonsteroidal anti-inflammatory drug, has historically been employed as an analgesic antipyretic agent. In recent investigations, it has also been established as an effective cardioprotective agent during ischemia/reperfusion, hypoxia/reoxygenation, exogenous peroxynitrite administration, and experimentally induced myocardial infarction (17, 18, 30–33, 39, 40, 50). We have previously reported both chronic and acute acetaminophen treatment (0.35 mM) to be cardioprotective following ischemia/reperfusion in the isolated perfused guinea pig myocardium (17, 18). High-performance liquid chromatography analysis shows that treatment with acetaminophen at this molarity yields arterial and venous concentrations of 45–50 µg/ml (44). These values fall within the range of therapeutic human plasma concentrations (10–100 µg/ml) and well below those concentrations resulting in hepatotoxicity (≥300 µg/ml; Refs. 21, 36, 37).

Additional studies from our laboratory have demonstrated that acute acetaminophen treatment also confers protection in a canine model of myocardial infarction (18, 33). However, while structural, functional, and biochemical evidence of acetaminophen-mediated cardioprotection exists, mechanistic data are noticeably absent. It is presently believed that the mechanism of action may involve antioxidant properties of this drug conveyed by its phenolic structure; however, additional work is required to further delineate the pathway for this observed protection (18, 31, 32).

The goal of the present study was to examine the mechanistic pathway by which acetaminophen mediates cardioprotection. We found that, in our model, ischemia/reperfusion causes a significant increase in mitochondrial permeability transition pore (MPTP) opening and mitochondrial cytochrome c release, and that acetaminophen treatment during injury significantly and completely blocks these effects. In addition, we report that ischemia/reperfusion causes a significant increase in late stage apoptotic myocytes in both vehicle- and acetaminophen-treated hearts. However, although acetaminophen significantly decreases late stage apoptotic myocytes compared with vehicle-treated hearts following injury, its inhibition was not complete. The incomplete attenuation of late stage apoptosis in response to treatment with acetaminophen implies that, although this drug is successful at completely inhibiting MPTP opening and cytochrome c release, additional pathways of apoptosis are still active in the ischemic/reperfused Langendorff heart. These results confirm prior reports of acetaminophen-mediated cardioprotection and suggest a mechanistic pathway to explain this protection following ischemia/reperfusion.


   MATERIALS AND METHODS
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 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals and Langendorff preparation. Hartley strain male guinea pigs (400 ± 25 g) were obtained from Elm Hill Laboratories (Wilmington, MA) and allowed a minimum of 3 days to acclimate to their new environment. Following Institutional Animal Care and Use Committee review and approval of study protocols, guinea pigs were anesthetized using isoflurane in accordance with National Institutes of Health and United States Department of Agriculture guidelines.

Hearts were isolated and perfused in situ via the cannulated aorta and subsequently extracted and attached to a Langendorff perfusion apparatus as previously described (9, 10, 30). Pacing electrodes were placed at the base of the right ventricle to control heart rate at ~200 beats/min (model no. S44; Grass-Telefactor, West Warwick, RI), and physiological heart temperature was monitored using a thermistor probe (model no. BAT-12; Physitemp, Clifton, NJ). Coronary perfusion pressure was controlled hydrostatically (55 ± 5 mmHg).

Perfusate and ischemia/reperfusion protocol. Hearts were perfused with a modified Krebs-Henseleit physiological salt solution/buffer (KHB) containing (in mM): 128.0 NaCl, 4.7 KCl, 1.5 MgSO4·7H2O, 2.5 CaCl2, 1.2 KH2PO4, 24.9 NaHCO3, 10.0 glucose, and 2.0 pyruvate, with 200 µU/ml insulin. Perfusate was warmed to 37°C, equilibrated with a 95% O2-5% CO2 gas mixture (pH 7.40 ± 0.02), and delivered from a water-jacketed perfusion reservoir. Flow was allowed to vary naturally and was continuously monitored ultrasonically (model no. T106 flow meter; Transonic Systems, Ithaca, NY).

Guinea pigs were randomly assigned to vehicle (KHB) or acetaminophen (0.35 mM dissolved in KHB) treatment groups. Following extraction and suspension from the Langendorff apparatus, all hearts remained untreated and were perfused with KHB for the first 15 min of the baseline stabilization period. Subsequently, hearts were treated with either acetaminophen or vehicle (added to the perfusate reservoir) for the remainder of the 30-min baseline period and for the duration of the Langendorff perfusion. Low-flow global myocardial ischemia (1 ml/min) was then induced for 30 min followed by 60 min of reperfusion. Animal choice and age and the use of low-flow ischemia were employed to be consistent with previous reports from our laboratory establishing the functional cardioprotective capacity of acetaminophen (31, 32).

Monitored variables included heart rate (HR; beats/min), coronary perfusate flow (CPF; ml·min–1·g–1), and coronary perfusion pressure (CPP; mmHg). A data acquisition system (model no. 214; iWorx/CB Sciences, Dover, NH) in series with a personal computer (Compaq Evo running LabScribe software version 6.0) was used to record monitored variables. Metabolic data including pH, PO2 (mmHg), and PCO2 (mmHg) were recorded using a standard blood-gas analyzer (model no. 248; Chiron Diagnostics, Norwood, MA).

Myocardial homogenization and fractionation. Hearts were randomly divided into vehicle and acetaminophen treatment groups and exposed to the ischemia/reperfusion protocol described above. Monitored variables and metabolic data were collected just before perfusion termination (i.e., at 15 min of baseline for control hearts or the end of reperfusion). Following termination of Langendorff perfusion, hearts were crushed and immersed in homogenization buffer (10 ml/g) containing (in mM): 210.0 mannitol, 7.0 sucrose, and 5.0 4-morpholinopropanesulfonic acid, pH 7.4, 37°C, 1 tablet/10 ml buffer protease inhibitor tablets (complete mini; Roche Diagnostics, Indianapolis, IN). Hearts were then homogenized using both polytron blade (model no. PT 2100; Kinematica, Littau-Lucerne, Switzerland) and Teflon (model no. JR4000; Arrow Engineering, Hillside, NJ) homogenizers. Separation of cytosolic and mitochondrial fractions was modified from previously described procedures (6, 46). The homogenate was centrifuged at 1,000 g for 10 min at 4°C, and the resulting supernatant was centrifuged at 7,000 g for 10 min at 4°C. The pellet from the second centrifugation represented the mitochondrial fraction and was resuspended in 10 mM sodium phosphate, pH 9.0. The supernatant represented the cytosolic fraction. An additional group of hearts was homogenized following 15 min of baseline perfusion as a control.

Mitochondrial swelling. Mitochondrial suspensions were assayed spectrophotometrically (540 nm) at 25°C for changes in light scattering (8, 15, 25, 41). Mitochondrial fractions of heart homogenate were assessed following ischemia/reperfusion from both vehicle- and acetaminophen-treated hearts following a Bradford assay to determine total protein concentration. Light absorbance values were expressed as a percentage with respect to the average of baseline hearts.

Myofibrillar ultrastructure. A separate group of hearts was used to assess myofibrillar ultrastructure as previously described (18). Hearts were randomly assigned to one of two termination groups (15-min baseline or reperfusion) corresponding to the experimental period following which the Langendorff perfusion would be terminated and the heart subjected to fixation. The reperfusion group was further divided into either vehicle or acetaminophen treatment groups.

Hearts were perfused with Karnovsky's fixative for 2 min at the end of baseline or reperfusion conditions. Hearts were then submerged in fixative, and 2- to 3-mm3 blocks of myocardium were removed longitudinally from the anterior free wall of the left ventricle midway between the left ventricular and left anterior descending branches of the left main coronary artery, equidistant from base to apex. Blocks were subsequently fixed using 1% osmium tetroxide and dehydrated in graded ethanol (17, 40). Samples were embedded in Epon-Araldite cocktail, sectioned with a diamond knife ultramicrotome (model no. LKB-2088; LKB, Bromma, Sweden), and viewed with an electron microscope (model no. JEM-100CXII; JEOL USA, Peabody, MA), using standard protocols (3).

Mitochondrial cytochrome c release. Following termination of Langendorff perfusion, hearts were freeze-clamped in liquid nitrogen using a modified Wollenberger clamp and stored at –80°C until homogenization (39). A Bradford assay (Bio-Rad Protein Assay; Bio-Rad, Hercules, CA) was used to determine total protein concentration. Cytosolic and mitochondrial fractions from baseline and vehicle- and acetaminophen-treated ischemic/reperfused heart homogenates were then loaded randomly into wells (i.e., the investigator was blinded for band density quantification). Proteins were resolved on a 15% SDS polyacrylamide gel and transferred to a polyvinylidene difluoride membrane. The membrane was then probed with a mouse monoclonal antibody to cytochromec(clone 7H8.2C12, 1:1,000; Stressgen Bioreagents, Victoria, BC, Canada). Cytosolic and mitochondrial fractions were further probed for rabbit anti-{alpha}-actin (1:2,500; Sigma-Aldrich, St. Louis, MO) and rabbit anti-voltage-dependent anion channel/Porin (VDAC, 1:2,500; Sigma-Aldrich), respectively, as loading controls. Film was scanned, and quantification was carried out through optical density analysis using Scion image software.

Isolation of ventricular myocytes and fluorescence-activated cell sorting. Hearts were randomly divided into vehicle and acetaminophen treatment groups and exposed to the ischemia/reperfusion protocol described above. Hemodynamic and metabolic data were collected at 15-min baseline, 30-min ischemia, and 60-min reperfusion. Isolation of ventricular myocytes was a modification of previously described methods (24, 35). Briefly, following ischemia/reperfusion, hearts were perfused with calcium-free KHB for 2–3 min to arrest contractions. Hearts were then perfused with 0.08% collagenase type 2 (Worthington Biochemical, Lakewood, NJ) dissolved in calcium-free KHB in a recirculating mode for ~10–15 min. Subsequently, hearts were removed from the perfusion apparatus, and ventricles were cut longitudinally into six to eight slices and incubated and mildly agitated with 15 ml of KHB plus 0.08% collagenase at 37°C for 5 min. Cells were centrifuged at 10,000 g for 50 s and washed two times in KHB. An additional group of hearts was digested following 15 min of baseline perfusion as a control.

Following isolation, myocytes were resuspended in annexin V binding buffer, loaded with annexin V-fluorescein isothiocyanate (FITC) and propidium iodide (PI), and analyzed using a fluorescence-activated cell sorter (FACS model no. FC500 flow cytometer; Beckman Coulter, Fullerton, CA) according to the manufacturer's protocol (Vybrant Apoptosis Assay Kit no. 3; Molecular Probes, Carlsbad, CA). Early and late stage apoptotic myocytes were characterized as annexin V-FITC or both annexin V-FITC and PI positive, respectively.

Statistics. Reported values are means ± SE. Data were analyzed using an ANOVA followed by Tukey's multiple comparison test (InStat; GraphPad, San Diego, CA). Significance was accepted at P < 0.05 in all cases.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
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 REFERENCES
 
Hemodynamic and metabolic parameters. Hemodynamic and metabolic data were collected following 15-min baseline, ischemia, and reperfusion for hearts subjected to myocyte isolation and just before perfusion termination (following baseline or reperfusion) for hearts subjected to freeze-clamping and homogenization. There were no significant differences between vehicle- and acetaminophen-treated hearts or between baseline and reperfused hearts during any sample time. Expected hemodynamic differences in CPF were observed between baseline and ischemic hearts in the myocyte isolation studies (Table 1).


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  		Table 1. Hemodynamic and metabolic data during myocardial ischemia/reperfusion

 
Acetaminophen treatment inhibits mitochondrial swelling following myocardial ischemia/reperfusion. Decreases in light absorbance are representative of increases in mitochondrial matrix volume as a result of the opening of MPTPs, subsequent water influx, and mitochondrial swelling (27, 42). In the present study, we examined differences in light absorbance of isolated mitochondria following baseline and ischemia/reperfusion as an index of MPTP opening. Mitochondrial and cytosolic fractions were each probed for voltage-dependent anion channel (VDAC), a mitochondrial outer membrane protein, to confirm mitochondrial membrane integrity. The lack of VDAC in the cytosolic fraction of vehicle-treated baseline heart homogenates, compared with its presence in mitochondrial fractions, demonstrates that mitochondrial membranes were intact (Fig. 1A). Our spectrophotometric results indicate a significant decrease in mitochondrial light absorbance of vehicle-treated ischemic/reperfused mitochondrial fractions (0.66 ± 0.04) compared with either baseline or postreperfusion acetaminophen-treated mitochondrial fractions (1.27 ± 0.09). However, there were no significant changes in light absorbance between acetaminophen-treated mitochondrial fractions following ischemia/reperfusion and baseline values, suggesting that acetaminophen treatment attenuates ischemia/reperfusion-induced mitochondrial swelling via inhibition of the MPTP (Fig. 1B).


Figure 1
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  		Fig. 1. Spectrophotometric analysis of mitochondrial swelling. A: Western blot showing the lack of voltage-dependent anion channel (VDAC) in the cytosolic fraction of a representative vehicle-treated baseline heart following homogenization and centrifugation. B: hearts were homogenized, and mitochondria were isolated via centrifugation following baseline and ischemia/reperfusion in vehicle- and acetaminophen (APAP)-treated hearts (n = 4/group). Mitochondrial swelling was measured as a decrease in light scattering at 540 nm (absorbance at 540 nm; A540) and expressed as a percentage of the average corresponding baseline value (A540 baseline) (protein density ~1.0 µg/µl). V, vehicle-treated hearts; A, APAP-treated hearts. *P < 0.05, as determined by ANOVA followed by Tukey's multiple comparison test, compared with corresponding baseline hearts. {dagger}P < 0.05, as determined by ANOVA followed by Tukey's multiple comparison test, compared with ischemic/reperfused vehicle-treated hearts.

 
To assess whether acetaminophen preserves myofibrillar ultrastructure during cardiac ischemia/reperfusion, we examined electron micrographs in both vehicle- and acetaminophen-treated hearts following injury. As shown in Fig. 2, myofibrillar ultrastructure from vehicle-treated hearts displayed extensive postreperfusion tissue damage compared with myocardial sections from either baseline or acetaminophen-treated hearts. As indicated in Fig. 2, mitochondria from left ventricular free wall sections appear dense and intact in baseline and acetaminophen-treated ischemic/reperfused hearts. However, mitochondria from vehicle-treated ischemic/reperfused hearts are visually swollen and structurally more rounded. These data further support the conclusion that acetaminophen treatment inhibits MPTP-induced mitochondrial swelling following ischemia/reperfusion.


Figure 2
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  		Fig. 2. Electron micrograph analysis of left ventricle free wall. Representative electron micrographs following baseline (A), vehicle ischemia/reperfusion (B), and APAP ischemia/reperfusion (C) (n = 2/group). Swollen mitochondria (white arrows in B relative to A and C) imply the opening of mitochondrial permeability transition pores. The presence of APAP during ischemia/reperfusion appears to attenuate permeability transition and, consequently, mitochondrial swelling. Note the similarity in mitochondrial color and shape between baseline (A) and APAP-treated ischemic/reperfused hearts (C).

 
Molecular consistency between vehicle-treated hearts. Previous studies from our laboratory that have examined the effects of acetaminophen following cardiac injury have reported mostly descriptive and functional data (17, 18, 30, 31, 40). Drug- and vehicle-treated hearts were considered similar if the hemodynamic and metabolic parameters collected at baseline were not statistically different. In the present study, we further explored the molecular consistency between discrete Langendorff preparations at baseline by comparing cytosolic cytochromeccontent. We found quantitative consistency in cytosolic cytochromeccontent between vehicle-treated hearts following 15 min of baseline perfusion (Fig. 3A). These data show the first evidence that our preparations are biochemically consistent.


Figure 3
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  		Fig. 3. Western blot analysis of cytosolic and mitochondrial cytochrome c heart homogenate fractions following ischemia/reperfusion. Hearts were freeze-clamped, homogenized, and separated into mitochondrial and cytosolic fractions following the experimental perfusion. Approximately 10 µg of protein from each heart were resolved on a 15% SDS polyacrylamide gel and probed for cytochrome c and the appropriate loading control (either {alpha}-actin or VDAC) by Western blotting. A: hearts were freeze-clamped and homogenized immediately following 15 min of baseline perfusion with Krebs-Henseleit buffer (n = 4). Ratio of cytosolic cytochrome c (Cytc) to {alpha}-actin band intensity was 0.76 ± 0.03 in baseline hearts. B–D: hearts were freeze-clamped and homogenized immediately following 15 min of baseline perfusion or following ischemia/reperfusion in vehicle- and APAP-treated hearts (n = 4/group). B: representative Western blots from cytosolic (cyt) and mitochondrial (mito) heart homogenate fractions. C: statistical analysis of cytosolic cytochrome c content normalized to {alpha}-actin and baseline. D: statistical analysis of mitochondrial cytochrome c content normalized to VDAC and baseline. V, vehicle-treated hearts; A, APAP-treated hearts. *P < 0.05, as determined by ANOVA followed by Tukey's multiple comparison test, compared with corresponding baseline hearts. {dagger}P < 0.05, as determined by ANOVA followed by Tukey's multiple comparison test, compared with ischemic/reperfused vehicle-treated hearts.

 
Acetaminophen treatment inhibits mitochondrial cytochrome c release following myocardial ischemia/reperfusion. Mitochondrial cytochrome c release is a known trigger for the intrinsic apoptotic cascade (49). In the present study, we analyzed cytosolic and mitochondrial cytochrome c content following baseline and ischemia/reperfusion. Our results indicate a significant increase in cytochrome c release from mitochondria to the cytosol following ischemia/reperfusion in vehicle-treated hearts (Fig. 3, B and C). In addition, therapeutic concentrations of acetaminophen, administered beginning at 15 min of baseline and continuing throughout the ischemia/reperfusion protocol, result in a significant and complete attenuation of cytochrome c release compared with vehicle-treated hearts. Cytosolic cytochrome clevels (normalized to {alpha}-actin and baseline hearts) were 11.15 ± 2.18 and 1.21 ± 0.48 in vehicle- and acetaminophen-treated hearts, respectively. No differences were noted between mitochondrial or cytosolic cytochrome c content of acetaminophen-treated hearts compared with corresponding baseline samples (Fig. 3, B–D). These data show that acetaminophen treatment significantly and completely inhibits the mitochondrial cytochrome c release normally observed following myocardial ischemia/reperfusion.

Acetaminophen treatment attenuates the number of late stage apoptotic myocytes following myocardial ischemia/reperfusion. Ischemia/reperfusion injury can induce both necrotic and apoptotic cell death (23). Our group has previously reported that acetaminophen mediates attenuation of necrotic cell death following myocardial infarction (33); however, the specific role of acetaminophen in myocardial apoptosis has not yet been explored. To address whether inhibition of apoptosis plays a role in the mechanism of acetaminophen-mediated cardioprotection, we isolated ventricular myocytes following baseline and ischemia/reperfusion. We then loaded myocytes with annexin V-FITC and PI and analyzed fluorescent intensity using flow cytometry. As shown in Fig. 4, the total percentage of late apoptotic cells following ischemia/reperfusion was significantly reduced in acetaminophen- vs. vehicle-treated hearts (58 ± 1 vs. 81 ± 5%, respectively). However, no significant differences were noted between treatment groups during early apoptosis (17 ± 5 vs. 17 ± 6% for vehicle- and acetaminophen-treated hearts, respectively). Additionally, significant increases in late stage apoptotic myocytes were observed in both treatment groups following reperfusion compared with data from baseline hearts. These data suggest that our preparation was successful at inducing late stage apoptosis and that acetaminophen may play a cardioprotective role by attenuating the progression of apoptosis in cardiomyocytes following ischemia/reperfusion.


Figure 4
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  		Fig. 4. Fluorescence-activated cell sorting (FACS) analysis of post-ischemic/reperfused ventricular myocytes. Hearts were digested with collagenase, and myocytes were isolated and loaded with annexin V-FITC and propidium iodide following 15-min baseline or ischemia/reperfusion in vehicle- and APAP-treated hearts (n = 4/group). Flow cytometry was used to determine percentage of early (A) and late stage (B) apoptotic myocytes in vehicle- and APAP-treated hearts following ischemia/reperfusion. C: representative FACS analysis of vehicle-treated ischemic/reperfused heart. J1, necrotic cells; J2, late apoptotic cells; J3, viable cells; J4, early apoptotic cells; B, baseline hearts; V, vehicle-treated hearts; A, APAP-treated hearts. *P < 0.05, as determined by ANOVA followed by Tukey's multiple comparison test, compared with myocytes from baseline hearts in the same stage of apoptosis. {dagger}P < 0.05, as determined by ANOVA followed by Tukey's multiple comparison test, compared with myocytes from vehicle-treated hearts in the same stage of apoptosis.

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
With the drastic rise in heart disease, the need for preventative cardiac care has become essential. Many groups have investigated the protective capacity of various compounds in inhibiting myocardial ischemia/reperfusion-induced injury. Varga et al. (48) investigated the effects of pretreatment with dexamethasone, a potent glucocorticoid, on post-ischemia/reperfusion. They reported that dexamethasone inhibits ventricular fibrillation via attenuation of mitochondrial cytochrome c release. Studies by Kovacs et al. (28) used nonspecific caspase administration at the onset of reperfusion to maintain cardiac function and limit both infarct size and apoptosis. Additional reports from Das et al. (12) examined the cardioprotective effects of pretreatment with palm tocotrienol, a vitamin E isomer, following myocardial ischemia/reperfusion. They demonstrated that treatment with tocotrienols derived from a tocotrienol-rich fraction of palm oil results in the ability to attenuate ischemia/reperfusion-induced damage via inhibition of c-Src phosphorylation and maintenance of proteasomal activity.

In this study, we examined the mechanistic basis for reported acetaminophen-mediated functional cardioprotection. Previous studies show that, in an in vivo canine preparation of myocardial infarction, acetaminophen treatment results in a significant reduction of necrotic tissue (33). In the present study, we proposed that acetaminophen might also have an effect on the mitochondrial pathway of apoptosis following ischemia/reperfusion. Specifically, we explored whether therapeutic concentrations of acetaminophen were able to attenuate MPTP opening, cytochromec release, and apoptotic cell death. The major finding of our study is that, following myocardial ischemia/reperfusion, acetaminophen treatment completely blocks opening of the MPTP and mitochondrial swelling as well as cytochrome c release from mitochondria. Furthermore, although acetaminophen attenuates late stage apoptosis, it does not completely block it. These results suggest that acetaminophen inhibits the MPTP-induced pathway of apoptosis; however, other pathways leading to apoptosis may not be affected by acetaminophen.

During a procedure to reestablish blood supply to an ischemic myocardium, one of the main concerns is the oxidative stress that occurs because of the formation of oxygen radicals and other reactive oxygen species (ROS; Refs. 2, 14, 47). When accompanied by additional postreperfusion environmental conditions, including mitochondrial matrix calcium overload, adenine nucleotide depletion, and elevated phosphate concentrations, permeability transition pores switch to the open conformation, thus triggering the intrinsic apoptotic cascade (19). Although endogenous antioxidant systems are capable of minimizing ROS-induced tissue injury under physiological conditions, they are insufficient to neutralize ROS following ischemia/reperfusion insult (4, 38). Previous reports demonstrate the efficacy of exogenously administered antioxidants in preventing myocardial ischemia/reperfusion-related injury in the Langendorff-perfused heart, including inhibition of MPTP opening (1, 5, 22, 26, 45).

Acetaminophen, when taken at therapeutic concentrations, has been established as a safe antipyretic and analgesic drug (36). More recently, this compound has also been established as an effective cardioprotective agent during myocardial ischemia/reperfusion injury (19, 30–32). Mechanistically, the phenolic hydroxyl group of acetaminophen likely donates its hydrogen atom to aid in the reported reduction of free radicals, namely peroxynitrite and hydroxyl radicals, postreperfusion (30–32, 36). Ischemia/reperfusion-induced oxidative stress is a well-known trigger for MPTP opening, mitochondrial cytochrome c release, and downstream apoptotic cell death pathway activation (16, 19, 34, 49). We hypothesize that acetaminophen-mediated inhibition of ROS generation results, in part, in attenuation of reperfusion-induced myocardial injury via a reduction in MPTP opening, mitochondrial cytochrome c release, and apoptotic cell death. Although some investigators report both cytochromec release and mitochondrial permeability transition during the ischemic period, many others reserve this method of damage for the reperfusion period (7, 19, 20, 29). Still, it is possible that the length and degree of ischemic insult play a crucial part in the onset of pore opening and mechanism of damage (11).

Acetaminophen-mediated inhibition of mitochondrial swelling and MPTP opening following ischemia/reperfusion. Reports indicate that mitochondrial swelling is indicative of MPTP opening and ultimately results in outer mitochondrial membrane rupture (13). Increases in mitochondrial swelling, as assessed by decreases in light absorbance, would therefore imply downstream cytochrome c release and activation of the mitochondrial-mediated pathway of apoptosis (13, 27). We found a significant decrease in the light absorbance of isolated mitochondria from vehicle-treated ischemic/reperfused hearts compared with either baseline, and acetaminophen treatment completely reversed this effect (Fig. 1B). These results suggest that our model of ischemia/reperfusion (i.e., 30 min of low-flow global ischemia and 60 min of reperfusion) successfully induced mitochondrial permeability pore opening at the completion of reperfusion, and that the presence of acetaminophen resulted in inhibition of this opening (Fig. 1B). These data are further strengthened by electron micrograph analysis showing preserved myofibrillar ultrastructure and intact mitochondria in acetaminophen-treated hearts, similar to baseline controls, and visually swollen mitochondria post-ischemia/reperfusion in vehicle-treated hearts (Fig. 2). These data suggest that acetaminophen completely attenuated pore opening following ischemia/reperfusion in our model.

Acetaminophen-mediated inhibition of mitochondrial cytochrome c release following ischemia/reperfusion. Following ischemia/reperfusion-induced outer mitochondrial membrane rupture in response to MPTP opening and mitochondrial swelling, cytochrome cis released into the cytosol to initiate the intrinsic pathway of apoptosis (19). We found a significant increase in cytosolic cytochrome c content, with a concomitant decrease in mitochondrial cytochrome c content, following ischemia/reperfusion in vehicle-treated hearts. This suggests that our model of ischemia/reperfusion was successful at inducing mitochondrial cytochrome c release at the completion of reperfusion. In addition, acetaminophen treatment resulted in a significant and complete inhibition of cytochrome c release following injury compared with vehicle-treated hearts. These data suggest that acetaminophen treatment completely inhibits mitochondrial cytochrome c release following ischemia/reperfusion in our model. We have shown (Figs. 1 and 2) that the observed inhibition of cytochrome c release is likely a response to the complete upstream inhibition of MPTP opening; however, it is possible that acetaminophen also exhibits functional cardioprotection via other pathways upstream of cytochrome c release.

Acetaminophen-mediated attenuation of late stage apoptosis following ischemia/reperfusion. In our protocol, early stage apoptotic myocytes were defined as those cells that were stained with annexin V-FITC. This population was composed of myocytes that had externalized phosphatidylserine residues and active caspases but no DNA degradation or loss of membrane integrity. Late stage apoptotic myocytes were defined as those cells that were both annexin V-FITC and PI positive. This myocyte population had active caspases and permeabilized cell membranes (43). We found that acetaminophen treatment significantly inhibited the number of late stage apoptotic myocytes compared with vehicle-treated hearts at the completion of reperfusion. However, there was also a significant increase in late stage apoptotic myocytes between baseline and acetaminophen-treated ischemic/reperfused hearts. This increasing index of damage following ischemia/reperfusion in acetaminophen-treated hearts was not apparent as mitochondrial swelling or cytochrome c release. It is possible that the changes in apoptotic cell death noted between treatment groups at reperfusion are due, in part, to acetaminophen-mediated MPTP inhibition and cytochrome c release. However, these data also suggest that while acetaminophen may abolish permeability pore transition and cytochrome c release following ischemia/reperfusion, other pathways of apoptosis, unaffected by acetaminophen treatment, are still active during injury. We propose that acetaminophen-mediated cardioprotection is, at least in part, specific to inhibition of MPTP-induced cytochrome c release and apoptosis.

Damaging postreperfusion oxidants, including peroxynitrite and hydroxyl radical, activate the intrinsic pathway of apoptosis, and the efficacy of acetaminophen in attenuating these compounds makes it a likely inhibitor of this pathway (31). However, it is possible that the incomplete attenuation of apoptosis in response to treatment is the result of ischemia/reperfusion-induced activation of other apoptotic cell death pathways, including the extrinsic pathway of apoptosis. Figure 5 is a schematic of the proposed mechanism of acetaminophen-mediated cardioprotection following ischemia/reperfusion insult and a model of our findings.


Figure 5
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  		Fig. 5. Mechanism of APAP-mediated cardioprotection. A: summary of study findings as they relate to the mechanism of APAP-mediated cardioprotection following ischemia/reperfusion. APAP completely inhibits mitochondrial permeability transition pore (MPTP) opening and mitochondrial cytochrome c release and partially attenuates apoptosis compared with vehicle-treated hearts following ischemia/reperfusion. ns, No significance. *P < 0.05. B: schematic of proposed mechanism of action of APAP following myocardial ischemia/reperfusion. Our studies imply that although APAP completely inhibits MPTP opening and mitochondrial cytochrome c release, apoptosis is not completely blocked. Thus additional apoptotic pathways are still active following insult.

 
These data begin to explain the mechanism of previous reports of acetaminophen-mediated cardioprotection following ischemia/reperfusion. They suggest that administration of acetaminophen just before an ischemic attack can result in attenuation of functional damage via inhibition of MPTP opening and cytochrome c release-induced apoptotic cell death following ischemia/reperfusion. Although these data are promising, in that they offer a historically safe alternative to preventative cardiac care, additional pathway details still need to be elucidated before clinical application.


   GRANTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was funded in part by Johnson and Johnson Corporate Office of Science and Technology/McNeil Consumer Specialty Products (to G. F. Merrill) and American Heart Association Grant-in-Aid No. 0555801T (to B. L. Firestein).


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. F. Merrill, 604 Allison Road, Piscataway, NJ 08854 (e-mail: Merrill{at}biology.rutgers.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Adlam VJ, Harrison JC, Porteous CM, James AM, Smith RA, Murphy MP, Sammut IA. Targeting an antioxidant to mitochondria decreases cardiac ischemia-reperfusion injury. FASEB J 19: 1088–1095, 2005.[Abstract/Free Full Text]
  2. Ambrosio G, Tritto I. Reperfusion injury: experimental evidence and clinical implications. Am Heart J 138: S69–S75, 1999.[CrossRef][Web of Science][Medline]
  3. Bazzola JJ, Russel LD. Electron Microscopy: Principles and Techniques for Biologists. Sudbury, MA: Jones and Bartlett, 1999.
  4. Blaustein A, Deneke SM, Stolz RI, Baxter D, Healey N, Fanburg BL. Myocardial glutathione depletion impairs recovery after short periods of ischemia. Circulation 80: 1449–1457, 1989.[Abstract/Free Full Text]
  5. Bognar Z, Kalai T, Palfi A, Hanto K, Bognar B, Mark L, Szabo Z, Tapodi A, Radnai B, Sarszegi Z, Szanto A, Gallyas F Jr, Hideg K, Sumegi B, Varbiro G. A novel SOD-mimetic permeability transition inhibitor agent protects ischemic heart by inhibiting both apoptotic and necrotic cell death. Free Radic Biol Med 41: 835–848, 2006.[CrossRef][Web of Science][Medline]
  6. Bopassa JC, Vandroux D, Ovize M, Ferrera R. Controlled reperfusion after hypothermic heart preservation inhibits mitochondrial permeability transition-pore opening and enhances functional recovery. Am J Physiol Heart Circ Physiol 291: H2265–H2271, 2006.[Abstract/Free Full Text]
  7. Borutaite V, Jekabsone A, Morkuniene R, Brown GC. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia. J Mol Cell Cardiol 35: 357–366, 2003.[CrossRef][Web of Science][Medline]
  8. Bosetti F, Baracca A, Lenaz G, Solaini G. Increased state 4 mitochondrial respiration and swelling in early post-ischemic reperfusion of rat heart. FEBS Lett 563: 161–164, 2004.[CrossRef][Web of Science][Medline]
  9. Bunger R, Haddy FJ, Gerlach E. Coronary responses to dilating substances and competitive inhibition by theophylline in the isolated perfused guinea pig heart. Pflügers Arch 358: 213–224, 1975.[CrossRef][Web of Science][Medline]
  10. Bunger R, Haddy FJ, Querengasser A, Gerlach E. An isolated guinea pig heart preparation with in vivo like features. Pflügers Arch 353: 317–326, 1975.[CrossRef][Web of Science][Medline]
  11. Chen Q, Moghaddas S, Hoppel CL, Lesnefsky EJ. Reversible blockade of electron transport during ischemia protects mitochondria and decreases myocardial injury following reperfusion. J Pharmacol Exp Ther 319: 1405–1412, 2006.[Abstract/Free Full Text]
  12. Das S, Powell SR, Wang P, Divald A, Nesaretnam K, Tosaki A, Cordis GA, Maulik N, Das DK. Cardioprotection with palm tocotrienol: antioxidant activity of tocotrienol is linked with its ability to stabilize proteasomes. Am J Physiol Heart Circ Physiol 289: H361–H367, 2005.[Abstract/Free Full Text]
  13. Di Lisa F, Bernardi P. Mitochondria and ischemia-reperfusion injury of the heart: fixing a hole. Cardiovasc Res 70: 191–199, 2006.[Abstract/Free Full Text]
  14. Ferrari R, Alfieri O, Curello S, Ceconi C, Cargnoni A, Marzollo P, Pardini A, Caradonna E, Visioli O. Occurrence of oxidative stress during reperfusion of the human heart. Circulation 81: 201–211, 1990.[Abstract/Free Full Text]
  15. Gadelha FR, Thomson L, Fagian MM, Costa AD, Radi R, Vercesi AE. Ca2+-independent permeabilization of the inner mitochondrial membrane by peroxynitrite is mediated by membrane protein thiol cross-linking and lipid peroxidation. Arch Biochem Biophys 345: 243–250, 1997.[CrossRef][Web of Science][Medline]
  16. Gateau-Roesch O, Argaud L, Ovize M. Mitochondrial permeability transition pore and postconditioning. Cardiovasc Res 70: 264–273, 2006.[Abstract/Free Full Text]
  17. Golfetti R, Rork T, Merrill G. Chronically administered acetaminophen and the ischemia/reperfused myocardium. Exp Biol Med (Maywood) 228: 674–682, 2003.[Abstract/Free Full Text]
  18. Golfetti R, VanDyke K, Rork T, Spiler N, Merrill G. Acetaminophen in the post-ischemia reperfused myocardium. Exp Biol Med (Maywood) 227: 1031–1037, 2002.[Abstract/Free Full Text]
  19. Halestrap AP, Clarke SJ, Javadov SA. Mitochondrial permeability transition pore opening during myocardial reperfusion–a target for cardioprotection. Cardiovasc Res 61: 372–385, 2004.[Abstract/Free Full Text]
  20. Halestrap AP, Connern CP, Griffiths EJ, Kerr PM. Cyclosporin A binding to mitochondrial cyclophilin inhibits the permeability transition pore and protects hearts from ischaemia/reperfusion injury. Mol Cell Biochem 174: 167–172, 1997.[CrossRef][Web of Science][Medline]
  21. Hardman JG, Limbird LL, Molinoff PB, Ruddon RW, Gilman AG. Goodman and Gilman's The Pharmacological Basis of Therapeutics. NewYork: McGraw-Hill, 1996.
  22. Hirai M, Hotta Y, Ishikawa N, Wakida Y, Fukuzawa Y, Isobe F, Nakano A, Chiba T, Kawamura N. Protective effects of EGCg or GCg, a green tea catechin epimer, against postischemic myocardial dysfunction in guinea-pig hearts. Life Sci 80: 1020–1032, 2007.[CrossRef][Web of Science][Medline]
  23. Honda HM, Korge P, Weiss JN. Mitochondria and ischemia/reperfusion injury. Ann NY Acad Sci 1047: 248–258, 2005.[CrossRef][Web of Science][Medline]
  24. Huang XD, Horackova M, Pressler ML. Changes in the expression and distribution of connexin 43 in isolated cultured adult guinea pig cardiomyocytes. Exp Cell Res 228: 254–261, 1996.[CrossRef][Web of Science][Medline]
  25. Hunter FE Jr, Gebicki JM, Hoffsten PE, Weinstein J, Scott A. Swelling and lysis of rat liver mitochondria induced by ferrous ions. J Biol Chem 238: 828–835, 1963.[Free Full Text]
  26. Javadov SA, Lim KH, Kerr PM, Suleiman MS, Angelini GD, Halestrap AP. Protection of hearts from reperfusion injury by propofol is associated with inhibition of the mitochondrial permeability transition. Cardiovasc Res 45: 360–369, 2000.[Abstract/Free Full Text]
  27. Kaasik A, Safiulina D, Zharkovsky A, Veksler V. Regulation of mitochondrial matrix volume. Am J Physiol Cell Physiol 292: C157–C163, 2007.[Abstract/Free Full Text]
  28. Kovacs P, Bak I, Szendrei L, Vecsernyes M, Varga E, Blasig IE, Tosaki A. Non-specific caspase inhibition reduces infarct size and improves post-ischaemic recovery in isolated ischaemic/reperfused rat hearts. Naunyn Schmiedebergs Arch Pharmacol 364: 501–507, 2001.[CrossRef][Web of Science][Medline]
  29. Lundberg KC, Szweda LI. Initiation of mitochondrial-mediated apoptosis during cardiac reperfusion. Arch Biochem Biophys 432: 50–57, 2004.[Web of Science][Medline]
  30. Merrill G, McConnell P, Vandyke K, Powell S. Coronary and myocardial effects of acetaminophen: protection during ischemia-reperfusion. Am J Physiol Heart Circ Physiol 280: H2631–H2638, 2001.[Abstract/Free Full Text]
  31. Merrill GF. Acetaminophen and low-flow myocardial ischemia: efficacy and antioxidant mechanisms. Am J Physiol Heart Circ Physiol 282: H1341–H1349, 2002.[Abstract/Free Full Text]
  32. Merrill GF, Goldberg E. Antioxidant properties of acetaminophen and cardioprotection. Basic Res Cardiol 96: 423–430, 2001.[CrossRef][Web of Science][Medline]
  33. Merrill GF, Rork TH, Spiler NM, Golfetti R. Acetaminophen and myocardial infarction in dogs. Am J Physiol Heart Circ Physiol 287: H1913–H1920, 2004.[Abstract/Free Full Text]
  34. Orrenius S, Gogvadze V, Zhivotovsky B. Mitochondrial oxidative stress: implications for cell death. Annu Rev Pharmacol Toxicol 47: 143–183, 2007.[CrossRef][Web of Science][Medline]
  35. Piper H, Isenberg G. (editors). Isolated Adult Cardiomyocytes. Boca Raton, FL: CRC, 1989.
  36. Prescott LF. Paracetamol (Acetaminophen): a Critical Bibliographic Review. New York: Taylor and Francis, 2001.
  37. Prescott LF. Paracetamol: past, present, future. Am J Ther 7: 143–147, 2000.[Medline]
  38. Przyklenk K, Kloner RA. Superoxide dismutase plus catalase improve contractile function in the canine model of the "stunned myocardium." Circ Res 58: 148–156, 1986.[Abstract/Free Full Text]
  39. Rork TH, Hadzimichalis NM, Kappil MA, Merrill GF. Acetaminophen attenuates peroxynitrite-activated matrix metalloproteinase-2-mediated troponin I cleavage in the isolated guinea pig myocardium. J Mol Cell Cardiol 40: 553–561, 2006.[CrossRef][Web of Science][Medline]
  40. Rork TH, Van Dyke K, Spiler NM, Merrill GF. Acetaminophen in the hypoxic and reoxygenated guinea pig myocardium. Exp Biol Med (Maywood) 229: 1154–1161, 2004.[Abstract/Free Full Text]
  41. Rousou AJ, Ericsson M, Federman M, Levitsky S, McCully JD. Opening of mitochondrial KATP channels enhances cardioprotection through the modulation of mitochondrial matrix volume, calcium accumulation, and respiration. Am J Physiol Heart Circ Physiol 287: H1967–H1976, 2004.[Abstract/Free Full Text]
  42. Ruiz-Meana M, Garcia-Dorado D, Pina P, Inserte J, Agullo L, Soler-Soler J. Cariporide preserves mitochondrial proton gradient and delays ATP depletion in cardiomyocytes during ischemic conditions. Am J Physiol Heart Circ Physiol 285: H999–H1006, 2003.[Abstract/Free Full Text]
  43. Schmid I, Uittenbogaart C, Jamieson BD. Live-cell assay for detection of apoptosis by dual-laser flow cytometry using Hoechst 33342 and 7-amino-actinomycin D. Nat Protoc 2: 187–190, 2007.[Medline]
  44. Spiler NM, Rork TH, Merrill GF. An old drug with a new purpose: cardiovascular actions of acetaminophen (paracetamol). Curr Drug Targets Cardiovasc Haematol Disord 5: 419–429, 2005.[CrossRef][Medline]
  45. Sztark F, Ichas F, Ouhabi R, Dabadie P, Mazat JP. Effects of the anaesthetic propofol on the calcium-induced permeability transition of rat heart mitochondria: direct pore inhibition and shift of the gating potential. FEBS Lett 368: 101–104, 1995.[CrossRef][Web of Science][Medline]
  46. Tokarska-Schlattner M, Zaugg M, da Silva R, Lucchinetti E, Schaub MC, Wallimann T, Schlattner U. Acute toxicity of doxorubicin on isolated perfused heart: response of kinases regulating energy supply. Am J Physiol Heart Circ Physiol 289: H37–H47, 2005.[Abstract/Free Full Text]
  47. Tortolani AJ, Powell SR, Misik V, Weglicki WB, Pogo GJ, Kramer JH. Detection of alkoxyl and carbon-centered free radicals in coronary sinus blood from patients undergoing elective cardioplegia. Free Radic Biol Med 14: 421–426, 1993.[CrossRef][Web of Science][Medline]
  48. Varga E, Nagy N, Lazar J, Czifra G, Bak I, Biro T, Tosaki A. Inhibition of ischemia/reperfusion-induced damage by dexamethasone in isolated working rat hearts: the role of cytochrome c release. Life Sci 75: 2411–2423, 2004.[CrossRef][Web of Science][Medline]
  49. Weiss JN, Korge P, Honda HM, Ping P. Role of the mitochondrial permeability transition in myocardial disease. Circ Res 93: 292–301, 2003.[Abstract/Free Full Text]
  50. Zhu YZ, Chong CL, Chuah SC, Huang SH, Nai HS, Tong HT, Whiteman M, Moore PK. Cardioprotective effects of nitroparacetamol and paracetamol in acute phase of myocardial infarction in experimental rats. Am J Physiol Heart Circ Physiol 290: H517–H524, 2006.[Abstract/Free Full Text]




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