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Effects of endothelin-1 on mitochondrial function during the protection against myocardial cell apoptosis
BBRC Biochemical and Biophysical Research Communications 305 (2003) 898–903 www.elsevier.com/locate/ybbrc
Effects of endothelin-1 on mitochondrial function during the protection against myocardial cell apoptosisq Eri Iwai-Kanai,a Koji Hasegawa,a,* Souichi Adachi,b Masatoshi Fujita,c Masaharu Akao,a Teruhisa Kawamura,a and Toru Kitaa a
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan b Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan c College of Medical Technology, Kyoto University, Kyoto, Japan Received 23 March 2003
Abstract Endothelin-1 is a potent survival factor against myocardial cell apoptosis. While apoptotic stimuli often perturb mitochondrial function by decreasing the membrane potential as well as oxygen consumption, it is unknown whether ET-1 can rescue such perturbation by apoptotic stimuli. Administration of endothelin-1 inhibited the H2 O2 -induced release of cytochrome c from mitochondria to the cytosol in cardiac myocytes, indicating the involvement of the mitochondria-dependent pathway in the anti-apoptotic effect of endothelin-1. We showed here by cytofluorimetric analysis that endothelin-1 prevented the H2 O2 -induced decrease of membrane potential. However, endothelin-1 was unable to reverse the H2 O2 -mediated decrease in oxygen consumption and electron transport in the mitochondria of cardiac myocytes. Endothelin-1 was unable to rescue cardiac myocytes from apoptosis when administered after the decrease in mitochondrial membrane potential. These data suggest that endothelin-1 does not target the mitochondrial respiratory chain, but rather stabilizes the mitochondrial membrane during the protection against apoptosis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Apoptosis; Endothelin-1; Cardiac myocyte; Mitochondrion
Endothelin-1 (ET-1) is a 21-residue peptide originally isolated from supernatants of vascular endothelial cells [1]. Elevation of cardiac and plasma ET-1 levels has been reported in acute myocardial infarction [2], hypertension [3], and heart failure [4]. Therefore, ET-1 may be involved in the pathophysiology of such disease states. ET-1 acts not only as a vasoconstrictive and growth-promoting peptide, but also as a survival factor against apoptosis [5,6]. This anti-apoptotic effect is mainly mediated through a type A-receptor dependent pathway that involves Gq protein. Subcellular signaling pathways for the ET type A receptor include phosphaq Abbreviations: ET-1, endothelin-1; H2O2, hydrogen peroxide; JC1, 5,50 ,6,60 -tetrachloro1,10 ,3,37-tetra-ethylbenzimidazolylcarbocyanine iodido; PI, propidium iodide; MAPK, mitogen-activated protein kinase; TUNEL, TdT-mediated dUTP-biotin nick end labeling. * Corresponding author. Fax: +81-75-751-3203. E-mail address: [email protected] (K. Hasegawa).
tidylinositol 3 kinase, mitogen-activated protein kinases [5], and calcineurin [6,7]-mediated pathways. While activation of these multiple subcellular signaling pathways appears to coordinately mediate protective effects of ET1, the critical step point at which ET-1 acts during the apoptotic process is unknown. The mitochondrion is an organelle that synthesizes ATP by oxidative phosphorylation through electron transport and contains a double membrane [8]. Using four multi-subunit complexes, I–IV, which reside in the inner membrane, four electrons are transferred at a time to molecular oxygen, producing two molecules of water. The proton gradient generated during this process accounts for the mitochondrial transmembrane potential. Finally, ATP is synthesized by the transport of these protons into the mitochondrial matrix through the mitochondrial Hþ -ATPase. Apoptotic stimuli often perturb mitochondrial function by decreasing the membrane potential as well as oxygen consumption [9–11]. It is
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00839-8
E. Iwai-Kanai et al. / Biochemical and Biophysical Research Communications 305 (2003) 898–903
unknown whether ET-1 can rescue such perturbation by apoptotic stimuli. In this study, we provide evidence that the protective effect of ET-1 against myocardial cell apoptosis occurs at the mitochondrial level. In the mitochondria of cardiac myocytes, ET-1 does not prevent the defect of respiratory function but can prevent the decrease in membrane potential. Thus, ET-1 may stabilize the mitochondrial membrane during the protection against apoptosis.
Materials and methods Cytofluorimetric analysis. To measure the mitochondrial membrane potential, cells were incubated for 10 min at 37 °C in culture medium containing 1 lM 5,50 ,6,60 -tetrachloro-1,10 ,3,37-tetra-ethylbenzimidazolylcarbocyanine iodide (JC-1) and 5 lg/mL propidium iodide (PI) (both from Molecular Probes), followed by analysis within 30 min of the addition of fluorochrome in a Becton–Dickinson FACScalibur cytofluorometer. After suitable compensation, fluorescence was recorded at different wavelengths: JC-1 at 525 nm and PI at 600 nm [12,13]. To detect the changes in mitochondrial oxygen consumption in cardiac myocytes, we used the intracellular oxidation of dihydrorhodamine 123 to the fluorescent compound rhodamine 123, as previously described [10,14]. Briefly, cells were incubated for 10 min at 37 °C in respiration buffer (0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgCl2 , 10 mM Kþ Hepes, 5 mM KH2 PO4 , 1 mM ADP, and 5 mM succinate, pH 7.4) containing digitonin (final concentration 0.005%) to allow permeation of the mitochondrial outer membrane. After making sure by trypan blue staining that 90% of the cells were broken, the cells were treated with dihydrorhodamine 123 for 20 min at room temperature, followed by analysis of fluorescence in a Becton– Dickinson FACScalibur cytofluorometer. Oxygen electrode measurements. The activity of mitochondrial electron transport complexes I–IV was measured using an oxygen electrode, as previously described [8,15]. The cells were harvested with trypsin, and then centrifuged and resuspended in respiration buffer (0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgCl2 , 10 mM Kþ Hepes, and 5 mM KH2 PO4 , pH 7.4). One-half milliliter of the
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suspension was injected into a chamber containing 2.5 ml of air-saturated respiration buffer with 1 mM ADP that had been prewarmed to 37 °C. The cells were permeabilized with digitonin (final concentration 0.005%), and substrates and inhibitors were added in the following order and at these final concentrations: malate, 5 mM pyruvate, 5 mM; rotenone, 100 nM; succinate, 5 mM; antimycin A, 50 nM; ascorbate, 1 mM; and TMPD, 0.4 mM. Where noted, the uncoupling agent KCN was added at a final concentration of 5 mM. The oxygen concentration was calibrated with air-saturated buffer, assuming 390 ng-atoms of oxygen/ml of buffer. Transient downward deflections in the figures indicate the points at which new reagents were added to the samples in the oxygen electrodes. Rates of azide-sensitive oxygen consumption are expressed as ng-atoms of oxygen/min/3 107 cells. Detection of DNA fragmentation. The cells were subjected to the terminal deoxynucleotidyl transfer-mediated end labeling of fragmented nuclei (TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay), as previously described [5,12,16]. Statistical analysis. Data are presented as means SE. Statistical comparisons were performed by the use of unpaired two-tailed StudentÕs t tests and a probability value of < 0:05 was taken to indicate significance.
Results ET-1 prevents the H2 O2 -induced decrease of mitochondrial membrane potential, but not that of mitochondrial oxygen consumption To examine changes in mitochondrial membrane potential and oxygen consumption by H2 O2 and ET-1, we performed cytofluorimetric analysis [12,13]. Cardiac myocytes were incubated with saline or H2 O2 in the presence or the absence of ET-1 for 24 h. As shown in Fig. 1, stimulation with H2 O2 did not alter cell membrane permeability, as indicated by the lack of increase in propidium iodide binding to DNA. However, the number of cells with low JC-1 (cells in lower left quadrant) increased in H2 O2 -treated cells compared with that in saline-stimulated cells, indicating that H2 O2 reduced
Fig. 1. ET-1 prevents H2 O2 -induced decrease of mitochondrial membrane potential cardiac myocytes were stimulated with H2 O2 (105 M) or ET-1 (107 M) as indicated for 24 h. The mitochondrial membrane potential of these cells was measured with a fluorescence-activated cell sorter. The Y-axis indicates cell membrane permeability shown by PI fluorescence and the X-axis represents mitochondrial membrane potential shown by potential sensitive JC-1 dye fluorescence. The number of cells with low JC-1 (cells in lower left quadrant), shown at the bottom of each panel, represents the mean SEM of three independent experiments.
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the mitochondrial membrane potential. ET-1 prevented this effect of H2 O2 and reduced the number of cells with low JC-1. These findings indicate that ET-1 prevented the H2 O2 -induced decrease of mitochondrial membrane potential. We assessed mitochondrial oxygen consumption by analyzing the oxidation of dihydrorhodamine 123 to the fluorescent compound rhodamine 123 with cytofluorimetry [14]. As shown in Fig. 2, stimulation of cardiac myocytes with H2 O2 increased the number of cells with low dihydrorhodamine 123 fluorescence (cells in M1 in Fig. 2), indicating that H2 O2 decreased mitochondrial oxygen consumption. Notably, in contrast to the data of JC1 (mitochondrial membrane potential), the number of cells with low dihydrorhodamine 123 was still high even in cells treated with ET-1 in addition to H2 O2 . These findings indicate that ET-1 does not prevent the H2 O2 -induced inhibition of mitochondrial oxygen consumption. Taken together, these data imply that ET-1 can prevent the H2 O2 -induced decrease of membrane potential, but not that of oxygen consumption in the mitochondria of cardiac myocytes. ET-1 does not reverse the H2 O2 -induced decrease in mitochondrial electron transport To further investigate the effect of ET-1 on mitochondrial functions related to the respiratory chain, the activity of mitochondrial electron transport was measured using an oxygen electrode [8,15]. Mitochondrial electron transport via each complex was assessed by oxygen consumption produced by a substrate specific to each complex.
The changes of the oxygen uptake in cardiac myocytes treated with H2 O2 and ET-1 were examined. As shown in Figs. 3A and B, the oxygen consumption generated by all of the tested substrates was lower in H2 O2 -stimulated cardiac myocytes than in saline-stimulated cells. These findings indicate that H2 O2 decreased the activity of mitochondrial electron transport in all four complexes. In particular, the decrease in the activity of Complexes III and IV was striking. The activity of these four complexes was still low even when cells were treated with ET-1 in addition to H2 O2 . These findings indicate that ET-1 is unable to prevent the H2 O2 -mediated decrease in mitochondrial electron transport. The critical time point of ET-1-mediated protection during the apoptotic process Finally, we investigated the critical no-return point at which ET-1 is unable to inhibit myocardial cell apoptosis. First, we examined the time-course of changes in mitochondrial membrane potential and oxygen consumption during H2 O2 -induced myocardial cell apoptosis by cytofluorimetric analysis, as described above. As shown in Fig. 4, the percentage of cells with low mitochondrial oxygen consumption was markedly increased 6 h after the stimulation with H2 O2 . At this stage, however, the percentage of cells with low mitochondrial membrane potential was similar to that of the cells at the 0 time point (just before stimulation). The percentage of cells with low membrane potential was increased 12 h after the stimulation. Thereafter, the percentage of cells with low mitochondrial oxygen consumption and that of cells with low mitochondrial membrane potential did not change. These findings demonstrate that the decrease in oxygen
Fig. 2. ET-1 does not prevent the H2 O2 -mediated decrease in mitochondrial oxygen consumption. Cardiac myocytes were stimulated with H2 O2 (105 M) or ET-1 (107 M) as indicated for 24 h. Mitochondrial oxygen consumption was estimated by measuring oxidation of dihydrorhodamine 123 using a fluorescence-activated cell sorter. The Y-axis indicates cell counts and the X-axis indicates mitochondrial oxygen consumption as indicated by the dihydrorhodamine fluorescence. The number of cells with low dihydrorhodamine (cells in M1), shown at the bottom of each panel, represents the mean SEM of three independent experiments.
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Fig. 4. The time-course of changes of mitochondrial function during H2O2-induced myocardial cell apoptosis. The time-course of changes of mitochondrial membrane potential and oxygen consumption during H2 O2 -induced myocardial cell apoptosis were examined by cytofluorimetric analysis. The closed triangles indicate % of cardiac myocytes with low mitochondrial oxygen consumption as shown by low DHR fluorescence, and the closed squares indicate % of cells with low mitochondrial membrane potential as shown by low JC-1 fluorescence. Values are means SE of three independent experiments. # and *, p < 0:0001 different from 0 time point. Fig. 3. ET-1 does not prevent the H2 O2 -induced defect in mitochondrial electron transport. The function of mitochondrial electron transport complexes I–IV in cardiac myocytes was assayed using an oxygen electrode cuvette. Numerical values represent azide-sensitive oxygen consumption (ng-atoms O2 /min). (A) Transient downward deflections indicate the points at which the substrates (described at the bottom of the panel) specific for various segments of the electron transport path were added to the samples in the oxygen electrodes. (B) Quantitative analysis of oxygen consumption produced by a substrate specific for each electron transport complex. Values are means SE of three independent experiments.
consumption precedes the decrease in membrane potential in the mitochondria of cardiac myocytes after stimulation with H2 O2 . Finally, we examined the critical time period within which ET-1 can rescue cardiac myocytes from H2 O2 induced apoptosis by quantitatively analyzing TUNELpositive cells. Cardiac myocytes were treated with H2 O2 for 48 h, during which ET-1 was administered at various time points. As shown in Fig. 5A, brown-stained TUNEL-positive cardiac myocytes were found in H2 O2 stimulated cells (upper panel). These TUNEL-positive cells also displayed apoptotic morphological features. Brown-staining may specifically indicate the presence of DNA fragmentation, since no staining was observed when we omitted terminal deoxytransferase treatment (middle panel). Simultaneous administration of ET-1 and H2 O2 decreased the H2 O2 -induced percentage of TUNEL-positive myocytes (lower panel). As shown in
Fig. 5B, when ET-1 was administered at 6 h after the stimulation with H2 O2 , it could still decrease the percentage of TUNEL-positive myocytes. However, when ET-1 was administered later than 12 h after H2 O2 stimulation, it could not decrease the percentage of TUNEL-positive myocytes induced by H2 O2 stimulation. This fact taken together with the above finding that mitochondrial membrane potential decreases at 12 h after H2 O2 stimulation indicates that ET-1 is unable to rescue cardiac myocytes from apoptosis after the decrease in mitochondrial membrane potential.
Discussion ET-1 is a potent survival factor against apoptosis whose expression is up-regulated in various cardiovascular diseases [2–4]. However, the critical step at which ET-1 acts during the apoptotic process is unknown. Major findings of this study include: (1) ET-1 blocks the decrease in mitochondrial membrane potential during myocardial cell apoptosis, (2) ET-1 does not reverse the decreases in mitochondrial oxygen consumption and electron transport, (3) ET-1 can protect cardiac myocytes from apoptosis even when administered after the decrease in mitochondrial oxygen consumption, and (4) ET-1 cannot rescue myocytes from apoptosis when administered after the decrease in mitochondrial membrane potential.
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Fig. 5. Effects of ET-1 administered at various time points in myocardial cell apoptosis. Neonatal cardiac myocytes were treated with H2 O2 (105 M) for 48 h, during which ET-1 (107 M) was administered at various time points as indicated. The solid bar indicates time of stimulation with H2 O2 and the hatched bar indicates that of ET-1. (A) Representative photographs of TUNEL staining of cardiac myocytes. Terminal deoxytransferase (TdT) was omitted in the middle photograph. (B) Quantitative analysis of TUNEL-positive cardiac myocytes. Percentages of TUNEL-positive myocytes were calculated by counting an average of 400–500 nuclei in each slide, as previously described (4, 14, and 23). Values are means SE of three independent experiments. p < 0:0001.
The heart is an organ that requires constant highenergy supply. A drop in cellular ATP content has been recognized as an early feature of cell death [17]. However, direct assays of the cellular ATP content of cells undergoing apoptosis usually show no degradation of ATP until very late in the process [18]. Indeed, ATP is required for downstream events in apoptosis [19]. These findings suggest that depletion of ATP due to a decrease in mitochondrial oxidative phosphorylation in the early stage of cell death is not a critical event that determines the occurrence of apoptosis [11]. The present study
demonstrated that ET-1 protects cardiac myocytes from apoptosis without rescuing mitochondrial respiratory dysfunction. In addition, ET-1 can inhibit apoptosis even when administered after the decrease in mitochondrial oxygen consumption. These results suggest that ET-1 does not target the mitochondrial respiratory chain during protection against apoptosis and support the idea that mitochondrial respiratory dysfunction does not always lead to apoptosis of cardiac myocytes. Collapse of the mitochondrial membrane potential indicates the opening of a large conductance channel known as the permeability transition pore [11,20]. Previous studies have suggested that this event represents a central irreversible check-point during the apoptotic process [21,22]. The present study demonstrated that ET-1 reverses the decrease in mitochondrial membrane potential during the protection against myocardial cell apoptosis. These results suggest that stabilization of the mitochondrial membrane is a mechanism of ET-1-mediated protection against myocardial cell apoptosis. This study also demonstrated that ET-1 is unable to rescue myocytes when administered after the decrease in mitochondrial membrane potential. These findings are compatible with the idea that the loss of mitochondrial transmembrane potential represents a no-return checkpoint during the apoptotic process [21,22]. However, different situations have been reported in other cell types [18,23,24]. For example, HL-60 cells undergo apoptosis with little or no reduction of mitochondrial membrane potential [14]. It should be further investigated whether ET-1 can act as a survival factor in such cell types as well. The question arises as to how ET-1 stabilizes the mitochondrial membrane in cardiac myocytes. Several studies suggest that permeability transition pore opening is regulated by bcl-2 family proteins [18,23,25]. Our previous report demonstrated that ET-1 induces cardiac expression of bcl-2 protein [6], which prevents permeability transition pore opening. Therefore, this may be one mechanism of ET-1-mediated stabilization of the mitochondrial membrane. However, the activity of bcl2-related proteins is regulated not only by the quantity of these proteins, but also by their subcellular localization, post-translational modification, etc. The precise mechanisms by which ET-1 prevents the decrease in mitochondrial membrane potential during protection against apoptosis should be further investigated. The heart pump function quickly fails when ATP is not efficiently synthesized through mitochondrial oxidative phosphorylation. These deficiencies in the mitochondrial respiratory chain are associated with the development of heart failure [17]. The present study demonstrated that ET-1 is unable to rescue defects in mitochondrial respiratory function despite its protective effects against apoptosis. However, care must be taken when data obtained with cultured neonatal cardiac myocytes are applied to the in vivo setting in the adult. Further studies are needed
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to determine the role of ET-1 in experimental animal models of myocardial cell apoptosis, such as ischemia– reperfusion or heart failure in vivo.
Acknowledgments This work was supported in part by grants to K.H. from the Ministry of Education, Science and Culture of Japan. We would like to thank Mr. N. Sowa for his excellent technical assistance and Dr. M. Kanai for his valuable scientific advice.
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[11] D.R. Green, J.C. Reed, Mitochondria and apoptosis, Science 281 (1998) 1309–1312. [12] E. Iwai-Kanai, K. Hasegawa, T. Sawamura, M. Fujita, T. Yanazume, S. Toyokuni, S. Adachi, Y. Kihara, S. Sasayama, Activation of lectin-like oxidized low-density lipoprotein rreceptor-1 induces apoptosis in cultured neonatal rat cardiac myocytes, Circulation 104 (2001) 2948–2954. [13] A. Mathur, Y. Hong, B.K. Kemp, A.A. Barrientos, J.D. Erusalimsky, Evaluation of fluorescent dyes for the detection of mitochondrial membrane potential changes in cultured cardiomyocytes, Cardiovasc. Res. 46 (1) (2000) 126–138. [14] L.J. van Pelt, R. van Zwieten, R.S. Weening, D. Roos, A.J. Verhoeven, B.G.J.M. Bolscher, Limitations on the use of dihydrorhodamine 123 for flow cytometric analysis of the neutrophil respiratory burst, J. Immun. Meth. 191 (1996) 187–196. [15] V.K. Mootha, M.C. Wei, K.F. Buttle, L. Scorrano, V. Panoutsakopoulou, C.A. Mannella, S.J. Korsmeyer, A reversible component of mitochondrial respiratory dysfunction in apoptosis can be rescued by exogenous cytochrome c, EMBO J. 20 (2001) 661–671. [16] E. Iwai-Kanai, K. Hasegawa, M. Araki, T. Kakita, T. Morimoto, S. Sasayama, a- and b-adrenergic pathways differentially regulate cell type-specific apoptosis in rat cardiac myocytes, Circulation 100 (1999) 305–311. [17] B. OÕRourke, Apoptosis, Circ. Res. 85 (1999) 880–883. [18] E. Bossy-Wetzel, D.D. Newmeyer, D.R. Green, Mitochondrial cytochrome c release in apoptosis occurs upstream of DEVDspecific caspase activation and independently of mitochondrial transmembrane depolarization, EMBO J. 17 (1998) 37–49. [19] Y. Eguchi, S. Shimizu, Y. Tsujimoto, Intracellular ATP levels determine cell death fate by apoptosis or necrosis, Cancer Res. 57 (1997) 1835–1840. [20] S.J. Martin, D.R. Green, Protease activation during apoptosis: death by a thousand cuts?, Cell 82 (1995) 349–352. [21] G. Kroemer, Mitochondrial implication in apoptosis. Towards an endosymbiont hypothesis of apoptosis evolution, Cell Death Differ. 4 (1997) 443–456. [22] N. Zamzami, P. Marchetti, M. Castedo, T. Hirsch, S.A. Susin, B. Masse, G. Kroemer, Inhibitors of permeability transition interfere with the disruption of the mitochondrial transmembrane potential during apoptosis, FEBS Lett. 384 (1996) 53–57. [23] M.G. Vander Heiden, N.S. Chandel, E.K. Williamson, P.T. Schumacker, C.B. Thompson, Bcl-xL regulates the membrane potential and volume homeostasis of mitochondria, Cell 91 (1997) 627–637. [24] D.M. Finucane, N.J. Waterhouse, G.P. Amarante-Mendes, T.G. Cotter, D.R. Green, Collapse of the inner mitochondrial transmembrane potential is not required for apoptosis of HL60 cells, Exp. Cell Res. 251 (1999) 166–174. [25] J. Xiang, D.T. Chao, S.J. Korsmeyer, BAX-induced cell death may not require interleukin 1 b-converting enzyme-like proteases, Proc. Natl. Acad. Sci. USA 93 (1996) 14559–14563.
Effects of endothelin-1 on mitochondrial function during the protection against myocardial cell apoptosisq Eri Iwai-Kanai,a Koji Hasegawa,a,* Souichi Adachi,b Masatoshi Fujita,c Masaharu Akao,a Teruhisa Kawamura,a and Toru Kitaa a
Department of Cardiovascular Medicine, Graduate School of Medicine, Kyoto University, Kyoto, Japan b Department of Pediatrics, Graduate School of Medicine, Kyoto University, Kyoto, Japan c College of Medical Technology, Kyoto University, Kyoto, Japan Received 23 March 2003
Abstract Endothelin-1 is a potent survival factor against myocardial cell apoptosis. While apoptotic stimuli often perturb mitochondrial function by decreasing the membrane potential as well as oxygen consumption, it is unknown whether ET-1 can rescue such perturbation by apoptotic stimuli. Administration of endothelin-1 inhibited the H2 O2 -induced release of cytochrome c from mitochondria to the cytosol in cardiac myocytes, indicating the involvement of the mitochondria-dependent pathway in the anti-apoptotic effect of endothelin-1. We showed here by cytofluorimetric analysis that endothelin-1 prevented the H2 O2 -induced decrease of membrane potential. However, endothelin-1 was unable to reverse the H2 O2 -mediated decrease in oxygen consumption and electron transport in the mitochondria of cardiac myocytes. Endothelin-1 was unable to rescue cardiac myocytes from apoptosis when administered after the decrease in mitochondrial membrane potential. These data suggest that endothelin-1 does not target the mitochondrial respiratory chain, but rather stabilizes the mitochondrial membrane during the protection against apoptosis. Ó 2003 Elsevier Science (USA). All rights reserved. Keywords: Apoptosis; Endothelin-1; Cardiac myocyte; Mitochondrion
Endothelin-1 (ET-1) is a 21-residue peptide originally isolated from supernatants of vascular endothelial cells [1]. Elevation of cardiac and plasma ET-1 levels has been reported in acute myocardial infarction [2], hypertension [3], and heart failure [4]. Therefore, ET-1 may be involved in the pathophysiology of such disease states. ET-1 acts not only as a vasoconstrictive and growth-promoting peptide, but also as a survival factor against apoptosis [5,6]. This anti-apoptotic effect is mainly mediated through a type A-receptor dependent pathway that involves Gq protein. Subcellular signaling pathways for the ET type A receptor include phosphaq Abbreviations: ET-1, endothelin-1; H2O2, hydrogen peroxide; JC1, 5,50 ,6,60 -tetrachloro1,10 ,3,37-tetra-ethylbenzimidazolylcarbocyanine iodido; PI, propidium iodide; MAPK, mitogen-activated protein kinase; TUNEL, TdT-mediated dUTP-biotin nick end labeling. * Corresponding author. Fax: +81-75-751-3203. E-mail address: [email protected] (K. Hasegawa).
tidylinositol 3 kinase, mitogen-activated protein kinases [5], and calcineurin [6,7]-mediated pathways. While activation of these multiple subcellular signaling pathways appears to coordinately mediate protective effects of ET1, the critical step point at which ET-1 acts during the apoptotic process is unknown. The mitochondrion is an organelle that synthesizes ATP by oxidative phosphorylation through electron transport and contains a double membrane [8]. Using four multi-subunit complexes, I–IV, which reside in the inner membrane, four electrons are transferred at a time to molecular oxygen, producing two molecules of water. The proton gradient generated during this process accounts for the mitochondrial transmembrane potential. Finally, ATP is synthesized by the transport of these protons into the mitochondrial matrix through the mitochondrial Hþ -ATPase. Apoptotic stimuli often perturb mitochondrial function by decreasing the membrane potential as well as oxygen consumption [9–11]. It is
0006-291X/03/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0006-291X(03)00839-8
E. Iwai-Kanai et al. / Biochemical and Biophysical Research Communications 305 (2003) 898–903
unknown whether ET-1 can rescue such perturbation by apoptotic stimuli. In this study, we provide evidence that the protective effect of ET-1 against myocardial cell apoptosis occurs at the mitochondrial level. In the mitochondria of cardiac myocytes, ET-1 does not prevent the defect of respiratory function but can prevent the decrease in membrane potential. Thus, ET-1 may stabilize the mitochondrial membrane during the protection against apoptosis.
Materials and methods Cytofluorimetric analysis. To measure the mitochondrial membrane potential, cells were incubated for 10 min at 37 °C in culture medium containing 1 lM 5,50 ,6,60 -tetrachloro-1,10 ,3,37-tetra-ethylbenzimidazolylcarbocyanine iodide (JC-1) and 5 lg/mL propidium iodide (PI) (both from Molecular Probes), followed by analysis within 30 min of the addition of fluorochrome in a Becton–Dickinson FACScalibur cytofluorometer. After suitable compensation, fluorescence was recorded at different wavelengths: JC-1 at 525 nm and PI at 600 nm [12,13]. To detect the changes in mitochondrial oxygen consumption in cardiac myocytes, we used the intracellular oxidation of dihydrorhodamine 123 to the fluorescent compound rhodamine 123, as previously described [10,14]. Briefly, cells were incubated for 10 min at 37 °C in respiration buffer (0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgCl2 , 10 mM Kþ Hepes, 5 mM KH2 PO4 , 1 mM ADP, and 5 mM succinate, pH 7.4) containing digitonin (final concentration 0.005%) to allow permeation of the mitochondrial outer membrane. After making sure by trypan blue staining that 90% of the cells were broken, the cells were treated with dihydrorhodamine 123 for 20 min at room temperature, followed by analysis of fluorescence in a Becton– Dickinson FACScalibur cytofluorometer. Oxygen electrode measurements. The activity of mitochondrial electron transport complexes I–IV was measured using an oxygen electrode, as previously described [8,15]. The cells were harvested with trypsin, and then centrifuged and resuspended in respiration buffer (0.25 M sucrose, 0.1% bovine serum albumin, 10 mM MgCl2 , 10 mM Kþ Hepes, and 5 mM KH2 PO4 , pH 7.4). One-half milliliter of the
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suspension was injected into a chamber containing 2.5 ml of air-saturated respiration buffer with 1 mM ADP that had been prewarmed to 37 °C. The cells were permeabilized with digitonin (final concentration 0.005%), and substrates and inhibitors were added in the following order and at these final concentrations: malate, 5 mM pyruvate, 5 mM; rotenone, 100 nM; succinate, 5 mM; antimycin A, 50 nM; ascorbate, 1 mM; and TMPD, 0.4 mM. Where noted, the uncoupling agent KCN was added at a final concentration of 5 mM. The oxygen concentration was calibrated with air-saturated buffer, assuming 390 ng-atoms of oxygen/ml of buffer. Transient downward deflections in the figures indicate the points at which new reagents were added to the samples in the oxygen electrodes. Rates of azide-sensitive oxygen consumption are expressed as ng-atoms of oxygen/min/3 107 cells. Detection of DNA fragmentation. The cells were subjected to the terminal deoxynucleotidyl transfer-mediated end labeling of fragmented nuclei (TdT-mediated dUTP-biotin nick-end labeling (TUNEL) assay), as previously described [5,12,16]. Statistical analysis. Data are presented as means SE. Statistical comparisons were performed by the use of unpaired two-tailed StudentÕs t tests and a probability value of < 0:05 was taken to indicate significance.
Results ET-1 prevents the H2 O2 -induced decrease of mitochondrial membrane potential, but not that of mitochondrial oxygen consumption To examine changes in mitochondrial membrane potential and oxygen consumption by H2 O2 and ET-1, we performed cytofluorimetric analysis [12,13]. Cardiac myocytes were incubated with saline or H2 O2 in the presence or the absence of ET-1 for 24 h. As shown in Fig. 1, stimulation with H2 O2 did not alter cell membrane permeability, as indicated by the lack of increase in propidium iodide binding to DNA. However, the number of cells with low JC-1 (cells in lower left quadrant) increased in H2 O2 -treated cells compared with that in saline-stimulated cells, indicating that H2 O2 reduced
Fig. 1. ET-1 prevents H2 O2 -induced decrease of mitochondrial membrane potential cardiac myocytes were stimulated with H2 O2 (105 M) or ET-1 (107 M) as indicated for 24 h. The mitochondrial membrane potential of these cells was measured with a fluorescence-activated cell sorter. The Y-axis indicates cell membrane permeability shown by PI fluorescence and the X-axis represents mitochondrial membrane potential shown by potential sensitive JC-1 dye fluorescence. The number of cells with low JC-1 (cells in lower left quadrant), shown at the bottom of each panel, represents the mean SEM of three independent experiments.
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the mitochondrial membrane potential. ET-1 prevented this effect of H2 O2 and reduced the number of cells with low JC-1. These findings indicate that ET-1 prevented the H2 O2 -induced decrease of mitochondrial membrane potential. We assessed mitochondrial oxygen consumption by analyzing the oxidation of dihydrorhodamine 123 to the fluorescent compound rhodamine 123 with cytofluorimetry [14]. As shown in Fig. 2, stimulation of cardiac myocytes with H2 O2 increased the number of cells with low dihydrorhodamine 123 fluorescence (cells in M1 in Fig. 2), indicating that H2 O2 decreased mitochondrial oxygen consumption. Notably, in contrast to the data of JC1 (mitochondrial membrane potential), the number of cells with low dihydrorhodamine 123 was still high even in cells treated with ET-1 in addition to H2 O2 . These findings indicate that ET-1 does not prevent the H2 O2 -induced inhibition of mitochondrial oxygen consumption. Taken together, these data imply that ET-1 can prevent the H2 O2 -induced decrease of membrane potential, but not that of oxygen consumption in the mitochondria of cardiac myocytes. ET-1 does not reverse the H2 O2 -induced decrease in mitochondrial electron transport To further investigate the effect of ET-1 on mitochondrial functions related to the respiratory chain, the activity of mitochondrial electron transport was measured using an oxygen electrode [8,15]. Mitochondrial electron transport via each complex was assessed by oxygen consumption produced by a substrate specific to each complex.
The changes of the oxygen uptake in cardiac myocytes treated with H2 O2 and ET-1 were examined. As shown in Figs. 3A and B, the oxygen consumption generated by all of the tested substrates was lower in H2 O2 -stimulated cardiac myocytes than in saline-stimulated cells. These findings indicate that H2 O2 decreased the activity of mitochondrial electron transport in all four complexes. In particular, the decrease in the activity of Complexes III and IV was striking. The activity of these four complexes was still low even when cells were treated with ET-1 in addition to H2 O2 . These findings indicate that ET-1 is unable to prevent the H2 O2 -mediated decrease in mitochondrial electron transport. The critical time point of ET-1-mediated protection during the apoptotic process Finally, we investigated the critical no-return point at which ET-1 is unable to inhibit myocardial cell apoptosis. First, we examined the time-course of changes in mitochondrial membrane potential and oxygen consumption during H2 O2 -induced myocardial cell apoptosis by cytofluorimetric analysis, as described above. As shown in Fig. 4, the percentage of cells with low mitochondrial oxygen consumption was markedly increased 6 h after the stimulation with H2 O2 . At this stage, however, the percentage of cells with low mitochondrial membrane potential was similar to that of the cells at the 0 time point (just before stimulation). The percentage of cells with low membrane potential was increased 12 h after the stimulation. Thereafter, the percentage of cells with low mitochondrial oxygen consumption and that of cells with low mitochondrial membrane potential did not change. These findings demonstrate that the decrease in oxygen
Fig. 2. ET-1 does not prevent the H2 O2 -mediated decrease in mitochondrial oxygen consumption. Cardiac myocytes were stimulated with H2 O2 (105 M) or ET-1 (107 M) as indicated for 24 h. Mitochondrial oxygen consumption was estimated by measuring oxidation of dihydrorhodamine 123 using a fluorescence-activated cell sorter. The Y-axis indicates cell counts and the X-axis indicates mitochondrial oxygen consumption as indicated by the dihydrorhodamine fluorescence. The number of cells with low dihydrorhodamine (cells in M1), shown at the bottom of each panel, represents the mean SEM of three independent experiments.
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Fig. 4. The time-course of changes of mitochondrial function during H2O2-induced myocardial cell apoptosis. The time-course of changes of mitochondrial membrane potential and oxygen consumption during H2 O2 -induced myocardial cell apoptosis were examined by cytofluorimetric analysis. The closed triangles indicate % of cardiac myocytes with low mitochondrial oxygen consumption as shown by low DHR fluorescence, and the closed squares indicate % of cells with low mitochondrial membrane potential as shown by low JC-1 fluorescence. Values are means SE of three independent experiments. # and *, p < 0:0001 different from 0 time point. Fig. 3. ET-1 does not prevent the H2 O2 -induced defect in mitochondrial electron transport. The function of mitochondrial electron transport complexes I–IV in cardiac myocytes was assayed using an oxygen electrode cuvette. Numerical values represent azide-sensitive oxygen consumption (ng-atoms O2 /min). (A) Transient downward deflections indicate the points at which the substrates (described at the bottom of the panel) specific for various segments of the electron transport path were added to the samples in the oxygen electrodes. (B) Quantitative analysis of oxygen consumption produced by a substrate specific for each electron transport complex. Values are means SE of three independent experiments.
consumption precedes the decrease in membrane potential in the mitochondria of cardiac myocytes after stimulation with H2 O2 . Finally, we examined the critical time period within which ET-1 can rescue cardiac myocytes from H2 O2 induced apoptosis by quantitatively analyzing TUNELpositive cells. Cardiac myocytes were treated with H2 O2 for 48 h, during which ET-1 was administered at various time points. As shown in Fig. 5A, brown-stained TUNEL-positive cardiac myocytes were found in H2 O2 stimulated cells (upper panel). These TUNEL-positive cells also displayed apoptotic morphological features. Brown-staining may specifically indicate the presence of DNA fragmentation, since no staining was observed when we omitted terminal deoxytransferase treatment (middle panel). Simultaneous administration of ET-1 and H2 O2 decreased the H2 O2 -induced percentage of TUNEL-positive myocytes (lower panel). As shown in
Fig. 5B, when ET-1 was administered at 6 h after the stimulation with H2 O2 , it could still decrease the percentage of TUNEL-positive myocytes. However, when ET-1 was administered later than 12 h after H2 O2 stimulation, it could not decrease the percentage of TUNEL-positive myocytes induced by H2 O2 stimulation. This fact taken together with the above finding that mitochondrial membrane potential decreases at 12 h after H2 O2 stimulation indicates that ET-1 is unable to rescue cardiac myocytes from apoptosis after the decrease in mitochondrial membrane potential.
Discussion ET-1 is a potent survival factor against apoptosis whose expression is up-regulated in various cardiovascular diseases [2–4]. However, the critical step at which ET-1 acts during the apoptotic process is unknown. Major findings of this study include: (1) ET-1 blocks the decrease in mitochondrial membrane potential during myocardial cell apoptosis, (2) ET-1 does not reverse the decreases in mitochondrial oxygen consumption and electron transport, (3) ET-1 can protect cardiac myocytes from apoptosis even when administered after the decrease in mitochondrial oxygen consumption, and (4) ET-1 cannot rescue myocytes from apoptosis when administered after the decrease in mitochondrial membrane potential.
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Fig. 5. Effects of ET-1 administered at various time points in myocardial cell apoptosis. Neonatal cardiac myocytes were treated with H2 O2 (105 M) for 48 h, during which ET-1 (107 M) was administered at various time points as indicated. The solid bar indicates time of stimulation with H2 O2 and the hatched bar indicates that of ET-1. (A) Representative photographs of TUNEL staining of cardiac myocytes. Terminal deoxytransferase (TdT) was omitted in the middle photograph. (B) Quantitative analysis of TUNEL-positive cardiac myocytes. Percentages of TUNEL-positive myocytes were calculated by counting an average of 400–500 nuclei in each slide, as previously described (4, 14, and 23). Values are means SE of three independent experiments. p < 0:0001.
The heart is an organ that requires constant highenergy supply. A drop in cellular ATP content has been recognized as an early feature of cell death [17]. However, direct assays of the cellular ATP content of cells undergoing apoptosis usually show no degradation of ATP until very late in the process [18]. Indeed, ATP is required for downstream events in apoptosis [19]. These findings suggest that depletion of ATP due to a decrease in mitochondrial oxidative phosphorylation in the early stage of cell death is not a critical event that determines the occurrence of apoptosis [11]. The present study
demonstrated that ET-1 protects cardiac myocytes from apoptosis without rescuing mitochondrial respiratory dysfunction. In addition, ET-1 can inhibit apoptosis even when administered after the decrease in mitochondrial oxygen consumption. These results suggest that ET-1 does not target the mitochondrial respiratory chain during protection against apoptosis and support the idea that mitochondrial respiratory dysfunction does not always lead to apoptosis of cardiac myocytes. Collapse of the mitochondrial membrane potential indicates the opening of a large conductance channel known as the permeability transition pore [11,20]. Previous studies have suggested that this event represents a central irreversible check-point during the apoptotic process [21,22]. The present study demonstrated that ET-1 reverses the decrease in mitochondrial membrane potential during the protection against myocardial cell apoptosis. These results suggest that stabilization of the mitochondrial membrane is a mechanism of ET-1-mediated protection against myocardial cell apoptosis. This study also demonstrated that ET-1 is unable to rescue myocytes when administered after the decrease in mitochondrial membrane potential. These findings are compatible with the idea that the loss of mitochondrial transmembrane potential represents a no-return checkpoint during the apoptotic process [21,22]. However, different situations have been reported in other cell types [18,23,24]. For example, HL-60 cells undergo apoptosis with little or no reduction of mitochondrial membrane potential [14]. It should be further investigated whether ET-1 can act as a survival factor in such cell types as well. The question arises as to how ET-1 stabilizes the mitochondrial membrane in cardiac myocytes. Several studies suggest that permeability transition pore opening is regulated by bcl-2 family proteins [18,23,25]. Our previous report demonstrated that ET-1 induces cardiac expression of bcl-2 protein [6], which prevents permeability transition pore opening. Therefore, this may be one mechanism of ET-1-mediated stabilization of the mitochondrial membrane. However, the activity of bcl2-related proteins is regulated not only by the quantity of these proteins, but also by their subcellular localization, post-translational modification, etc. The precise mechanisms by which ET-1 prevents the decrease in mitochondrial membrane potential during protection against apoptosis should be further investigated. The heart pump function quickly fails when ATP is not efficiently synthesized through mitochondrial oxidative phosphorylation. These deficiencies in the mitochondrial respiratory chain are associated with the development of heart failure [17]. The present study demonstrated that ET-1 is unable to rescue defects in mitochondrial respiratory function despite its protective effects against apoptosis. However, care must be taken when data obtained with cultured neonatal cardiac myocytes are applied to the in vivo setting in the adult. Further studies are needed
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to determine the role of ET-1 in experimental animal models of myocardial cell apoptosis, such as ischemia– reperfusion or heart failure in vivo.
Acknowledgments This work was supported in part by grants to K.H. from the Ministry of Education, Science and Culture of Japan. We would like to thank Mr. N. Sowa for his excellent technical assistance and Dr. M. Kanai for his valuable scientific advice.
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