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1Institut National de la Santé et de la Recherche Médicale E 0226, Université Claude Bernard Lyon I and 2Hospices Civils, Lyon, France
Submitted 17 February 2005 ; accepted in final form 21 June 2005
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ABSTRACT |
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 TOP ABSTRACT MATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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lpr mice; cyclosporin A
, neurohormonal factors such as angiotensin II, NO, or mechanical stress. A complex network of signaling pathways transduce these diverse apoptotic stimuli in cardiomyocytes that include mainly the death receptors and the mitochondria (6). After Fas ligand binding, Fas receptors undergo trimerization and recruit Fas-associated death domain (FADD). Fas/FADD complex binds to the initiator caspase 8. According to the cell type, activated caspase 8 may propagate the apoptotic signal either through a direct activation of executioner downstream caspases or via the release of cytochrome c by mitochondria.
The involvement of mitochondria in apoptotic processes has been clearly demonstrated (15). Early after induction of apoptosis, a loss of the mitochondrial membrane potential, 
m, can be observed together with the opening of permeability transition pores in the inner mitochondrial membrane. Opening of this nonspecific megachannel results in the release of cytochrome c into the cytosol and subsequent proteolytic activation of the executioner caspases 3, 6, and 7. According to the ongoing metabolic status of the cell at the time of transition pore opening, including ATP and inorganic phosphate concentration, Ca2+ concentration, and production of oxygen-derived free radicals, cell death may be either apoptotic or necrotic (14).
Which of the Fas/Fas ligand (Fas/FasL) or the mitochondria pathway has a predominant role in cardiomyocyte death after a severe ischemic insult remains largely unknown. This is an important issue because both pathways are tightly regulated, and development of pharmacological agents targeting either signaling cascade is a clinically relevant issue. We combined here the genetic inactivation of the Fas receptor, on the one hand, to the pharmacological inhibition of the mitochondrial permeability transition pore (mPTP), on the other hand, to investigate which of the Fas or the mitochondria pathway might be predominantly activated during a prolonged ischemia-reperfusion in mice.
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MATERIALS AND METHODS |
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Eight- to 10-wk-old C57BL/6JICO (control) and C57BL/6-lpr (lpr) mice were purchased from Charles River laboratories (L'Arbresle, France) and CDTA-Centre National de la Recherche Scientifique (Orléans, France). C57BL/6-lpr mice display a loss-of-function mutation in the Fas gene (24). All animals had unrestricted access to water and food and were treated in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH Publication No. 85–23, revised 1996). The animal protocols used in this study were approved by the Veterinary Department of the Agriculture Agency of the French Government.
Myocardial Ischemia-Reperfusion Model
Eight- to 10-wk-old C57BL/6JICO and C57BL/6-lpr mice were anesthetized by intraperitoneal injection of 0.3 ml/10 g body wt of a 1:1 mixture of fentanyl citrate (0.011 mg/ml) and midazolam (0.4 mg/ml). Mice were orally intubated using a 22-gauge vinyl catheter and ventilated via a rodent ventilator (model 687, Harvard Apparatus) with a tidal volume of 0.2 ml and a breath rate of 160 breaths/min. Body temperature was monitored by a rectal thermometer and maintained at 36–37°C using a heating pad. After a midline sternotomy, a 7-0 silk suture was passed around the left anterior descending coronary artery (LAD), under a Zeiss microscope, for further creating ischemia and reperfusion. Ischemia was confirmed by ST segment elevation and the appearance of myocardial pallor. After 25 min of LAD occlusion, the silk snare was loosened, and reperfusion was confirmed by visual inspection and reduction of the ST segment shift on the ECG. The chest wall was closed with a 5-0 vicryl suture, and mice were allowed to recover in a temperature-controlled area. After 24 h of reperfusion, animals were reanesthetized, and hearts were harvested for further assessment of infarct size, myocardial apoptosis, and sensitivity of the mPTP to Ca2+ overload.
Experimental Protocol
At the end of the surgical preparation, mice were allowed 15 min for hemodynamic stabilization. Control and lpr mice were randomized to receive either saline or cyclosporin A (CsA). Thus the four following groups were constituted: control (C), control-CsA (C-CsA), lpr, and lpr-CsA. An additional group of sham-operated mice, that underwent no ischemia-reperfusion and did not receive any treatment, was used for caspase 3 activity measurements and assessment of Ca2+-induced mPTP opening.
Treatment by CsA
The mPTP inhibitor CsA (Sandimmun, Novartis) was injected into the peritoneal cavity at a dose of 40 mg/kg, three times a day. The first injection of CsA was performed 45 min before coronary artery occlusion.
Area at Risk and Infarct Size
At 24 h of reperfusion, the LAD was briefly reoccluded, and Unisperse blue dye (Ciba-Geigy) (0.25 ml of a 1% solution) was infused into the inferior vena cava to delineate the area at risk (19). Hearts were excised, and atrial and right ventricular tissues were trimmed off. The left ventricle was cut into four transverse slices that were weighed and photographed. Incubation in a 1% solution of 2,3,5-triphenyltetrazolium chloride (TTC) for 10 min at 32°C allowed differentiation between the viable (brick red) and necrotic (pale) myocardium. The slices were then rephotographed. Areas of the risk region and the infarcted myocardium were measured by computerized planimetry (ImageJ and Adobe Photoshop, 6.0 version). Infarct size and area at risk were calculated and expressed as percentage of the left ventricular (LV) weight.
mPTP Opening
Isolation of mitochondria. At the end of the experiment, the heart was harvested, and myocardium from the risk region was excised. From this sample of myocardium, mitochondria were isolated by homogenization and differential centrifugations in isolation buffer A (in mM): 70 sucrose, 210 mannitol, 1 EDTA in 50 Tris·HCl, pH 7.4. The homogenate was centrifuged twice at 1,300 g for 3 min, and the supernatant was centrifuged at 10,000 g for 10 min. The mitochondrial pellet was washed and resuspended in buffer B (in mM): 70 sucrose, 210 mannitol in 50 Tris·HCl, pH 7.4. To obtain aliquots of 1 mg mitochondrial proteins, according to Gornall's procedure (9), it was necessary to pool three ischemic zones of three different mice.
Ca2+-induced mPTP opening. Isolated mitochondria (1 mg protein) were added in 500 µl of buffer C (in mM: 150 sucrose, 50 KCl, 2 KH2PO4, 5 succinic acid in 20 Tris·HCl, pH 7.4) within a Teflon chamber equipped with a Ca2+-selective microelectrode in conjunction with a reference electrode (7, 12). A Synchronie software allowed a continuous recording of extramitochondrial calcium variations. Mitochondria were gently stirred for 1.5 min of preincubation. After this preincubation period, injections of 10 µM CaCl2 were repeated every minute. As depicted in Fig. 1, each 10 µM CaCl2 injection causes a peak of extramitochondrial Ca2+ concentration. Then, extramitochondrial Ca2+ concentration spontaneously returns to near baseline level, as Ca2+ enters the mitochondrial matrix via the Ca2+ uniporter (2). After a given Ca2+ load, extramitochondrial Ca2+ concentration abruptly increases, indicative of mPTP opening (Fig. 1). The amount of Ca2+ required to trigger mPTP opening is used as an indicator of the susceptibility of mPTP to Ca2+ overload.
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Caspase 3 activity. Heart lysates (in buffer A) were centrifuged at 16,000 g for 45 min at 4°C, and the supernatant fractions were collected. Caspase 3 activity was determined using a fluorometric assay system with DEVD-7-amino-4-methylcoumarin (DEVD-AMC) (Bachem Biochimie, France) as fluorochrome-associated caspase specific substrate. Fluorescence was measured on a spectrofluorometer (Perkin Elmer LS-5) at an excitation wavelength of 342 nm and an emission wavelength of 441 nm after incubation at 37°C for 60 min. The quantity of released AMC was calculated by transposing the fluorescence measurement of each point onto a scale with purified AMC.
Western blot analysis of cytochrome c. Hearts were homogenized in buffer A (in mM: 70 sucrose, 210 mannitol, 1 EDTA in 50 Tris·HCl, pH 7.4). After a centrifugation at 100,000 g for 1 h at 4°C, the final supernatants (cytosolic fractions) were collected. Cytosolic proteins equivalent to 50 µg protein were separated by 15% SDS-PAGE and transferred to nitrocellulose membrane, blocked with 5% nonfat dried milk in PBS for at least 1 h, washed with PBS-Tween, and incubated with anti-cytochrome c (clone 7H8.2C12, catalog no. 556433, Pharmingen) in PBS-Tween for 12 h at 4°C. Blots were developed with secondary antibody (anti-mice, Jackson ImmunoResearch) diluted 1/1,000 in PBS-Tween. After blots were washed with PBS-Tween, they were developed with an enhanced chemiluminescence (Lumi Light kit, Roche) and exposed for autoradiography. For stripping, blots were incubated for 5 min into a buffer containing 0.2 M Tris·HCl, pH 6.8, 8% SDS, and 20 mM mercaptoethanol at 100°C. Blots were washed three times for 10 min in PBS-Tween. The amount of protein to be used for detection was normalized using the GAPDH protein as loading control. The semiquantitative expression of cytochrome c was determined using a computerized software package (ImageJ).
Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling staining. In an additional subset of experiments, we assessed cardiomyocyte apoptosis using Terminal deoxynucleotide transferase-mediated dUTP nick-end labeling (TUNEL; fluorimetric technique), as previously described (1). For each slide, five separate fields each containing approximately 150–200 cardiomyocytes, were analyzed by two independent observers using a x40 objective. Cardiomyocytes with green nuclear fluorescence were defined as TUNEL-positive cells. The mean percentage of TUNEL-positive cardiomyocytes was calculated and expressed as the number of TUNEL-positive cells relative to the total number of myocytes (nuclei).
Statistical Analysis
Results are expressed as means ± SE. For caspase 3 activity and mPTP opening, groups were compared using one-way ANOVA with Tukey's post hoc test. For analysis of infarct size, we used ANOVA with post hoc Tukey's test. We adjusted the treatment effect on infarct size of either Fas mutation or CsA administration on the size of the area at risk. A P value <0.05 was considered as indicative of a statistically significant difference.
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RESULTS |
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Fas Protein in lpr Mice
We determined that the Fas mutation was actually present in lpr mice. RT-PCR revealed that the spot for Fas is different in wild-type (WT) and lpr mice (Fig. 2). This indicates that the Fas protein is present but modified (to be inactive) in lpr mice. This is in agreement with the expected loss-of-function mutation in lpr mice resulting from a transcriptional defect caused by a retrotransposon insertion into the Fas gene. This insertion causes premature termination and aberrant splicing of the Fas antigen mRNA in lpr mice. Absence of appropriate function of the muted protein has been previously reported by Wu et al. (24).
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The Ca2+ overload required to open the mPTP averaged 102 ± 11 µM in the sham-operated group (Fig. 3). The Ca2+ overload was significantly decreased in the control group, averaging 63 ± 9 µM (P < 0.05 vs. sham). Lpr hearts displayed similar sensitivity to calcium overload as controls, with a Ca2+ overload required for mPTP opening of 58 ± 6 µM. In contrast, CsA treatment significantly increased the resistance of the mPTP to Ca2+ overload, in control (103 ± 2 µM) as well as in lpr (98 ± 15 µM) hearts [P < 0.01 vs. C, P = not significant (NS) vs. sham].
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Area at risk was comparable among groups, averaging 50 ± 2%, 61 ± 4%, 58 ± 3%, and 52 ± 4% of the LV weight in C, lpr, C-CsA, and lpr-CsA groups, respectively. Mean infarct size in lpr mice was not significantly different from that in controls, averaging 83 ± 3% vs. 77 ± 5% of the area at risk, respectively. Treatment with CsA limited infarct size in both groups compared with controls: 51 ± 5% in C-CsA and 35 ± 4% of the area at risk in lpr-CsA (P < 0.01 vs, control and lpr; P = NS between CsA-treated groups). As depicted in Fig. 4, data points for the control and lpr groups are located on similar regression line. In contrast, data points for CsA-treated lpr and control mice lie below the regression line of controls, indicating that for any size of the area at risk they developed significantly smaller infarcts.
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DEVD-specific caspase activation. Caspase-3 activity was dramatically increased in control, averaging 57 ± 18 pmol·min–1·mg–1 vs. 6 ± 2 pmol·min–1·mg–1 in sham-operated hearts (Fig. 5B). Lpr hearts also exhibited an increased caspase 3 activity (49 ± 5 pmol·min–1·mg–1) that was similar to that observed in controls. CsA treatment significantly reduced caspase 3 activity in both control and lpr hearts: 16 ± 4 and 19 ± 10 pmol·min–1·mg–1, respectively (P < 0.05 vs. C and lpr).
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TUNEL. The percentage of apoptotic nuclei averaged 13 ± 2% and 11 ± 4% in control and lpr hearts, respectively (P = NS)(Fig. 6). CsA treatment significantly reduced the number of apoptotic cardiomyocytes, which averaged 5 ± 3% and 5 ± 2% in C-CsA and lpr-CsA hearts, respectively (P < 0.05 vs. control).
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DISCUSSION |
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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Activation of the Fas/FasL system can trigger apoptosis in various cell types, including cardiomyocytes (18). Fas and FasL expression are enhanced in cardiomyocytes exposed to hypoxia (22). To investigate the role of Fas in lethal cardiomyocyte injury after a 25-min ischemic insult, we used lpr mice that lack a functional Fas receptor (8). Lpr mice did not develop smaller infarcts compared with controls, nor did they exhibit a reduced activation of caspase 3, a pivotal effector caspase in the apoptotic program of cell death (4).
These results differ from earlier observations by Lee et al. (16), who reported a 63% reduction of cardiomyocyte apoptosis and necrosis in MRL-Fas-deficient mice. Jeremias et al. (13) observed a reduction in apoptotic cardiomyocyte death after ischemia-reperfusion in C57BL6-lpr mice (13). In contrast, our findings are in agreement with those of Li et al. (17), who recently demonstrated that lpr mice (as well as gld mice, i.e., with a nonfunctioning Fas ligand) do not develop smaller infarcts but display a significant reduction of granulation tissue apoptosis, thereby suggesting that the Fas/FasL interaction in granulations tissue cells may be more significant than that in cardiomyocytes (17). We demonstrated here that lpr mice display a modified, not functional, Fas receptor in cardiomyocytes. As depicted in Fig. 2, the Fas is present but modified in lpr cardiomyocytes. Wu et al. (24) reported that this muted protein was not functional (24). After irreversible ischemic insult, neither infarct size nor myocardial apoptosis, assessed by TUNEL, caspase 3 activity, and cytochrome c release, were reduced in these lpr hearts at 24 h of reperfusion. In agreement with Li et al. (17), our results suggest that Fas-induced apoptosis is not essential for cell death in the first hours of reperfusion, yet it may be of importance for LV remodeling at a later stage.
We then sought to determine whether mitochondrial damage might explain lethal cardiomyocyte injury in our experimental model. After a prolonged ischemia-reperfusion, it has been demonstrated that mitochondria may be challenged either indirectly, via G protein-coupled receptors, activation of phospholipases C or D and sphingomyelinases that lead to production of the apoptosis messenger ceramide (6), or directly via massive production of reactive oxygen species and Ca2+ overload that occur mainly at the time of reflow. We hypothesized that induction of mitochondrial permeability transition, independent of cell death receptor activation, may be responsible for necrotic and apoptotic cardiomyocyte death after a prolonged ischemia-reperfusion. Ischemia results in progressive intracellular acidosis, decline in ATP, and cytosolic accumulation of Na+ and Ca2+. On reperfusion, abrupt correction of acidosis by activation of the Na+/H+ exchanger causes a massive accumulation of Ca2+ in the cytosol via the Na+/Ca2+ exchanger, and Ca2+ enters mitochondria through the Ca2+ uniporter. In addition, the sudden influx of oxygen into anoxic cardiomyocytes causes a rapid burst of oxygen-derived free radicals, mediated through the interaction of O2 with ubisemiquinone formed during ischemia as a result of the inhibition of the respiratory chain (3, 11). The combination of oxidative stress and high Ca2+ concentration in the mitochondrial matrix, together with depletion of adenine nucleotide levels and inorganic phosphate accumulation, provide ideal conditions for mPTP opening (10). We demonstrated here that control hearts developed large infarcts and significant apoptosis after ischemia-reperfusion. Mitochondria isolated from control hearts displayed a significant reduction in their resistance to Ca2+ overload and exhibited an early mPTP opening. This is in line with a previous report from our group in the in vivo rabbit heart (1). Involvement of the mPTP in cardiomyocyte death was further demonstrated by using its inhibitor CsA, which delayed Ca2+-induced mPTP opening and dramatically reduced necrosis and apoptosis (23). CsA is, however, not fully specific for the mPTP because via its binding to the cytosolic cyclophilin A, it may influence other mechanisms, mostly the calcineurin/nuclear factor of activated T-cells (NFAT) pathway. We, however, recently demonstrated in the rabbit model that the very specific mPTP inhibitor NIM811 (that only binds to the mitochondrial cyclophilin D) provides similar benefit (1). One may argue that presence of necrotic tissue within the area at risk may be a confounding factor when assessing function of isolated mitochondria. It may be postulated that isolation procedure may differentially select subpopulations of mitochondria in large vs. small infarcts. To address this issue, in a previous study, we assessed mPTP opening after a brief (nonlethal) ischemia (instead of a prolonged ischemic insult with consecutive irreversible damage) (1). We found that the effect of CsA was maintained even in the absence of necrosis, indicating that reduced mPTP opening is a cause rather than a consequence of infarct size reduction.
Crosstalk has yet been described between the Fas and the mitochondria pathways: in some cell types, mitochondria may be challenged secondary to activation of the Fas pathway. In such cases, ligation of death receptors, such as Fas, activates caspase 8. Enzymatically active caspase 8 cleaves BID, and the truncated protein tBID relocates to mitochondria where it favors transition pore opening and executioner caspases processing (20). The use of lpr mice helps one to understand whether this might occur in cardiomyocytes after ischemia-reperfusion. We observed that apoptotic death, as estimated by caspase 3 activation, cytochrome c release, and TUNEL, and necrotic death were as important in lpr as in control mice, which strongly suggests that the Fas pathway does not seem to play a predominant role in cardiomyocyte death in our experimental model. The fact that CsA dramatically reduced apoptosis and necrosis in lpr mice further indicates that direct activation of caspase 3 after Fas activation, independent of mitochondria (as in the so-called type I cells), is likely not relevant after ischemia-reperfusion in mice cardiomyocytes. In other words, it suggests that cardiomyocytes behave as type II cells (20). In contrast, Date et al. (5) demonstrated, using cultured neonatal rat cardiomyocytes, that simulated hypoxia-reoxygenation upregulated the mRNA levels of Fas and FasL, activated caspase 8, and increased cytochrome c release from mitochondria (5). Also, Z-IETD.fmk, a selective caspase 8 inhibitor, reduced cytochrome c release from mitochondria, inhibited the activities of caspases 9 and 3, and limited apoptotic cardiomyocyte death (5). The difference from our results may be due to the use of non-mature cells and/or cell culture conditions. In contrast, our results are in agreement with those of Scarabelli et al. (21), who demonstrated, by using the isolated rat heart model, that even after inhibition of caspase 8, release of cytochrome c (which reflects mPTP opening) and downstream activation of caspase 9 are seen during reperfusion, suggesting that mitochondrial damage and cardiomyocyte death is triggered independently of Fas activation.
In conclusion, the present study suggests that prolonged ischemia-reperfusion induce cardiomyocyte necrotic and apoptotic death via activation of mitochondrial transition pore opening, independently of Fas activation.
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FOOTNOTES |
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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.
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TOPABSTRACTMATERIALS AND METHODSRESULTSDISCUSSIONGRANTSREFERENCES |
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35 kDa. The lymphoproliferation spontaneous mutation (Faslpr) is caused by the insertion of the early transposable element ETn in the Fas gene that causes premature termination and aberrant splicing of the Fas antigen mRNA in lpr mice. wt, Wild-type; –C, negative control.
0.01 vs. untreated control. A: Western blot for cytochrome c release. B: bar graph for caspase 3 activity.