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Estradiol prevents release of cytochrome c from mitochondria and inhibits ischemia-induced apoptosis in perfused heart
Experimental Gerontology 41 (2006) 704–708 www.elsevier.com/locate/expgero
Estradiol prevents release of cytochrome c from mitochondria and inhibits ischemia-induced apoptosis in perfused heart Ramune Morkuniene a,b, Odeta Arandarcikaite a, Vilmante Borutaite a,c,* a
Institute for Biomedical Research, Kaunas University of Medicine, Kaunas, Lithuania b Department of Biochemistry, Kaunas University of Medicine, Kaunas, Lithuania c Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK Received 28 October 2005; received in revised form 17 February 2006; accepted 21 February 2006 Available online 3 April 2006
Abstract The study investigated whether estradiol can prevent release of cytochrome c from mitochondria and induction of apoptosis after 30 and 60 min stop-flow heart ischemia in Langendorff-perfused female rat hearts. Pre-perfusion of hearts with 100 nM 17b-estradiol prevented the loss of cytochrome c from mitochondria, its accumulation in cytosol, and inhibition of respiration during ischemia. Estradiol strongly reduced activation of caspase-3-like activity and decreased DNA strand breaks in the nuclei of cardiomyocytes (measured by TUNEL staining). The results show that 17b-estradiol prevents the ischemia-induced release of cytochrome c from mitochondria, subsequent inhibition of mitochondrial respiration, and inhibits caspase activation and apoptosis. Therefore, inhibition of the intrinsic, mitochondria-mediated apoptotic pathway may be one of the mechanisms by which estrogens protect the heart against ischemic damage. q 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Estrogens; Ischemia; Mitochondria; Respiration; Cytochrome c
1. Introduction Estrogens are known to exert beneficial effects on the cardiovascular system after ischemia/reperfusion. It has been shown that 17b-estradiol reduces the extent of irreversible myocardial injury, ventricular arrhythmias, infarct size, and such effects have been detected in both male and female rats hearts (Hale et al., 1996; Zhai et al., 2000). A number of mechanisms have been proposed to explain the cardioprotective actions of estradiol in the heart. These include classical genomic or non-genomic responses. Estrogens act primarily through nuclear estrogen receptors (ERa and ERb), which regulate gene expression leading to the control of a variety of cellular functions (Mendelsohn and Karas, 1999). The second mechanism involves membrane or cytosolic estrogen receptors leading to transcriptional changes indirectly through activation of protein kinase cascades (Liao, 2001). However, recent evidence indicates that estrogens can also exert acute, * Corresponding author. Address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. Tel.: C44 1223 333342; fax: C44 1223 333345. E-mail address: [email protected] (V. Borutaite).
0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.02.010
transcription-independent effects on cellular functions (Babiker et al., 2002). These non-transcriptional actions of estrogens in myocardial ischemia/reperfusion have been suggested to be due to stimulation of NO production, inhibition of myocardial calcium accumulation or due to the antioxidant properties of estrogens (Zhai et al., 2000; Sugishita et al., 2003). We have previously shown that estrogens can acutely protect isolated heart mitochondria from the loss of cytochrome c induced by high calcium (Morkuniene et al., 2002), which is considered as one of the damaging factors during heart ischemia/reperfusion (Jennings, 1984). It was also known that cytochrome c release from mitochondria is one of the earliest mitochondrial events during ischemia (Piper et al., 1985; Toleikis et al., 1989), which causes inhibition of ATP synthesis, activation of caspases and nuclear apoptosis (Borutaite et al., 2003). In the present study, we aimed to elucidate whether estrogens can prevent activation of the intrinsic, mitochondriamediated apoptotic pathway in ischemic heart by protecting mitochondria from cytochrome c release. 2. Materials and methods Hearts from 2 months old, female Wistar rats were used in experiments. The hearts were perfused on Langendorff perfusion system with Krebs–Henseleit solution (11 mM
R. Morkuniene et al. / Experimental Gerontology 41 (2006) 704–708
glucose, 118 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, 1.7 mM MgSO4 and 0.7 mM Na pyruvate, pH 7.2 at 37 8C). Hearts were washed for 5 min with Krebs–Henseleit solution, then 100 nM 17bestradiol was added and hearts were perfused for another 15 min. Control hearts were perfused for the same time but without estradiol. Coronary flow rate was 10–12 ml/min in control and estradiol-perfused hearts. After 15 min of perfusion with/without estradiol, stop-flow ischemia was induced for 30 or 60 min. Hearts were homogenized with a motor-driven teflon/glass homogenizer in the medium containing 180 mM KCl, 20 mM Tris–HCl, 1 mM EGTA, pH 7.3. Mitochondrial and cytosolic fractions were separated by differential centrifugation (5 min! 750g, 10 min!6800g, 30 min!10,000g). Mitochondrial respiration was measured with a Clarke-type oxygen electrode at 37 8C in 1 ml incubation buffer containing 110 mM KCl, 2.24 mM MgCl2, 10 mM Tris–HCl, 5 mM KH2PO4, 4 IU/ml creatine kinase, 50 mM creatine, 1 mM pyruvateC1 mM malate (pH 7.2) and 1 mM ATP as described in Borutaite et al. (2003). In some experiments mitochondrial respiration was measured in the presence of 30 mM exogenous cytochrome c. Total cytochrome c and cytochrome a content was determined in mitochondria solubilized with 1% Triton X-100 (w/v). Sodium hydrosulphite-reduced minus hydrogen-peroxide-oxidized absorption difference spectra were recorded with a Hitachi-557 spectrophotometer. Cytochrome c content was estimated by using the absorption difference at the wavelength pair 550/535 nm and 3Z14.5 mMK1 cmK1; for cytochrome a—wavelength pair 605/630 nm and 3Z 10.4 mMK1 cmK1 as described in Rieske (1967). Cytochrome c content in cytosolic fractions was detected using Quantikinine M rat/mouse Immunoassay ELISA kit (R&D Systems). Cytosolic fraction proteins were dissolved in 0.5% Triton X-100 and further procedures were performed according to manufacturer’s protocol. Activity of caspases was measured in cytosolic extracts using 0.1 mM z-DEVD-p-nitroanilide, a caspase-3 substrate, as described in Borutaite et al. (2003). The number of apoptotic cells was quantified by dUTP nick end labeling (TUNEL) using CardioTACS (R&D Systems) in situ apoptosis detection kit. Hearts were fixed in 10% formalin. The tissues were embedded in molten paraffin and frozen. All other procedures were performed according to the manufacturer’s protocol. The number of TUNEL-positive cardiomyocytes was counted in at least 10 different microscopic fields on each section containing approximately 150 cardiomyocytes in each. Data were expressed as meansGSE of 5–10 separate experiments and analyzed with ANOVA followed by Tukey’s multiple comparison test. 3. Results We investigated whether high physiological concentration of 17b-estradiol (100 nM) can prevent the release of
705
cytochrome c from mitochondria to cytosol during ischemia in perfused rat female hearts. As can be seen in Fig. 1A, content of cytochrome c in mitochondria decreased by 19 and 26% after 30 and 60 min of ischemia, respectively, compared to the control level. However, in mitochondria isolated from ischemic hearts pre-perfused with 100 nM estradiol the content of cytochrome c was not significantly different from control or controlCestradiol mitochondria (Fig. 1A). Ischemia or estradiol itself had no significant effect on the mitochondrial content of cytochrome a, an integral component of the inner membrane (data not shown) suggesting that there was no general degradation of mitochondrial proteins and the release was specific for cytochrome c. Sixty minutes ischemia-induced loss of cytochrome c from mitochondria was observed (though to a lesser extent) even when mitochondria were isolated in sucrose-based buffer known to reduce dissociation of cytochrome c from mitochondria with damaged outer membrane. And again, the loss of cytochrome c was completely prevented
Fig. 1. Estradiol prevents release of cytochrome c from heart mitochondria to cytosol during ischemia. (A) Mitochondrial content of cytochrome c. (B) Cytochrome c content in cytosolic fractions. Female rat hearts were perfused for 15 min with Krebs–Henseleit solution with or without 100 nM of 17bestradiol, then stop-flow ischemia was induced for 30 or 60 min. Content of cytochrome c in isolated mitochondria is expressed as nmol/mg of mitochondrial protein. Cytosolic cytochrome c content is expressed as nmol/mg of cytosolic protein. *, Statistically significant effect of ischemia (p!0.05) if compared to control; #, statistically significant effect of estradiol (p!0.05) if compared to treatment without estradiol in the same group (30 or 60 min ischemia).
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when hearts were treated with estradiol: cytochrome c levels were 0.572G0.022, 0.493G0.017 (p!0.05) and 0.568G 0.011 nmol/mg protein in control, 60 min ischemic and 60 min ischemic plus estradiol mitochondria. Concomitant with decrease of mitochondrial content of cytochrome c during ischemia there was accumulation of this protein in cytosols and this was prevented when hearts were perfused with estradiol before induction of ischemia (Fig. 1B). Cytochrome c content in cytosolic extracts prepared from 30 to 60 min ischemic hearts increased by 45 and 56%, respectively, compared to control, while in cytosolic extracts from ischemicCestradiol hearts there was no significant increase in cytochrome c concentration compared to control level. Some cytochrome c was always present in control cytosols with or without estradiol and this may be caused by some inevitable damage of mitochondria during homogenization procedure of heart tissue. Altogether these results show that estradiol can prevent ischemia-induced release of cytochrome c from mitochondria to the cytosol. Next we tested whether perfusion of the heart with estradiol can prevent ischemia-induced inhibition of mitochondrial respiration. As can be seen from Fig. 2A, mitochondrial state
3 respiration after 30 and 60 min ischemia was lower by 47 and 63% compared to control mitochondria oxidizing pyruvate and malate. However, in mitochondria from 30 to 60 min ischemic hearts pre-perfused with estradiol state 3 respiratory rate was higher by 23 and 31%, respectively, compared to the respiration of ischemia-damaged mitochondria in the absence of estradiol. Exogenous cytochrome c added to the mitochondrial incubation medium stimulated respiratory rate by 110% after 30 min ischemia and by 192% after 60 min ischemia compared with respiration of ischemia-damaged mitochondria without added cytochrome c (Fig. 2B). However, when hearts were perfused with estradiol, cytochrome c stimulated respiration only by 70 and 106% in mitochondria from 30 to 60 min ischemia, respectively. Exogenous cytochrome c stimulates respiration only if mitochondrial outer membrane is damaged and permeable to cytochrome c. Preparation of isolated mitochondria are always to some degree heterogenous in respect to intactness of mitochondrial outer membrane as part of mitochondria are inevitably damaged during isolation (Piper et al., 1985), and this may be the reason why added cytochrome c stimulated the respiration of control mitochondria. Thus the data suggest that inhibition of respiration after ischemia is partially due to the loss of cytochrome c from mitochondria, and that estradiol partially prevented ischemiainduced respiratory inhibition in part by protection of mitochondria from the loss of cytochrome c. It has been shown that prolonged heart ischemia causes activation of caspases and nuclear fragmentation (Borutaite et al., 2003). We investigated whether perfusion of the hearts with 17b-estradiol can prevent activation of caspases and subsequent nuclear apoptosis. Caspase-3-like, DEVD-cleaving activity was low in control, but increased significantly after 60 min of ischemia (Fig. 3A). Activity of caspases was significantly (though not completely) blocked when hearts were perfused with 17b-estradiol before induction of ischemia (Fig. 3A). To further characterize apoptosis in ischemic myocardium, we used the TUNEL method to determine cells with DNA strand breaks. As can be seen from Fig. 3B, cardiomyocytes with DNA strand breaks were practically not detectable in control hearts, however, the number of such cells increased 10 times during ischemia. Perfusion of hearts with estradiol substantially reduced the number of cardiomyocytes with DNA strand breaks after 60 min ischemia, which is consistent with reduced activity of caspases due to prevention of cytochrome c release from mitochondria after such treatment. 4. Discussion
Fig. 2. Estradiol protects mitochondrial respiration damaged by ischemia. (A) Respiration rate without exogenous cytochrome c. (B) Respiration rate in the presence of 30 mM exogenous cytochrome c. 1 mM pyruvateC1 mM malate were used as respiratory substrate. Other symbols are as in Fig. 1.
In the present study we have demonstrated that estradiol blocked ischemia-induced release of cytochrome c from heart mitochondria into cytosol, subsequent activation of caspases and apoptosis. Estradiol also prevented inhibition of respiration of ischemia-damaged heart mitochondria caused by loss of cytochrome c. Similarly, a recent study has reported that estradiol reduced cytochrome c translocation and minimized hippocampal damage caused by transient global ischemia in rat
R. Morkuniene et al. / Experimental Gerontology 41 (2006) 704–708
Fig. 3. Estradiol prevents ischemia-induced caspase activation and nuclear apoptosis. (A) Caspase-3-like, DEVD-cleaving activity in cytosolic fractions from control, 60 min ischemic or 60 min ischemic plus estradiol hearts; (B) DNA fragmentation in cardiomyocytes. Other symbols are as in Fig. 1. Total number of cardiomyocytes analyzed for DNA fragmentation varied from 4000 to 10,000 in each group.
brain (Bagetta et al., 2004). Therefore, part of the cardioprotective action of estradiol seems likely to be related to preventing mitochondrial loss of cytochrome c. The classical target of estrogens is the nucleus, however, recent studies indicate that exogenously added estrogens are also transported to mitochondria (Moats and Ramirez, 1998, 2000). In in vivo experiments on ovariectomized rats it has been shown that added estrogen translocated from plasma membrane to mitochondria rather than to nuclei in variety of tissues (Moats and Ramirez, 1998). Therefore, it is likely that perfusion of the hearts with estradiol may also lead to at least partial accumulation of this hormone in mitochondria where it can act on membranes directly or indirectly through estrogen receptors that were recently identified in mitochondria (Chen et al., 2004; Yang et al., 2004). In isolated mitochondria, exogenously added estradiol has been shown to inhibit mitochondrial permeability transition pore (PTP)-related release of cytochrome c from mitochondria induced by high calcium (Morkuniene et al., 2002). Elevation of intracellular calcium is known as a causative damaging factor during ischemia/reperfusion (Jennings, 1984), which besides other effects may induce opening of mitochondrial PTP. PTP
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opening at reperfusion after short times of myocardial ischemia has been documented (Griffiths and Halestrap, 1995), though some studies indicated PTP opening during prolonged ischemia alone (Borutaite et al., 2003; Nazareth et al., 1991). PTP can cause cytochrome c release from mitochondria due to swelling of the mitochondria and rupture of the outer membrane. Thus the protective effect of estradiol may be related to the inhibition of PTP. On the other hand, we cannot rule out the possibility that the protective effect of estradiol on mitochondria in the perfused heart may be indirect as several authors have shown that estradiol inhibits calcium accumulation in the cytosol during ischemia/reperfusion (Zhai et al., 2000), and this may reduce the probability of PTP pore opening during ischemia. Another potential mechanism of indirect action of estrogens on mitochondria preventing loss of cytochrome c may be related to the effect of the hormone on protein kinase cascades through its binding to estrogen receptor a. It has been demonstrated that estrogens can activate the phosphoinositol3 kinase (PI3K) and the protein kinase Akt (Liao, 2001; Ho and Liao, 2002; Patten et al., 2004), which may phosphorylate and inactivate the proapoptotic proteins Bad and Bax (Datta et al., 1997) known to induce release of cytochrome c from mitochondria leading to apoptotic cell death. Therefore, preservation of mitochondria from the loss of cytochrome c might be related to estradiol-ER-a complex induced protein kinase Akt activation resulting in modification of proapoptotic Bcl-2 family proteins. The possibility that estrogens inhibit Bax- or Bad-induced cytochrome c release from mitochondria needs to be investigated in further studies. In conclusion, the present study provides evidence that at high physiological concentrations estradiol protects heart mitochondria from ischemia-induced release of cytochrome c, subsequent inhibition of respiration, activation of caspases and DNA fragmentation, and this may be one of the mechanisms by which estrogens preserve myocardial cell viability during ischemia/reperfusion. Acknowledgements We thank Dr Guy Brown for critical reading of manuscript and fruitful discussion. This work was supported by Lithuanian State Science and Studies Foundation and the British Heart Foundation. References Babiker, F.A., De Windth, L.J., Eickels, M., Grohe, C., Meyer, R., Doevendans, P.A., 2002. Estrogenic hormone action in the heart: regulatory network and function. Cardiovasc. Res. 53, 709–719. Bagetta, G., Chiappetta, O., Amantea, D., Iannone, M., Rotiroti, D., Costa, A., Nappi, G., Corosaniti, M.T., 2004. Estradiol reduces cytochrome c translocation and minimizes hippocampal damage caused by transient global ischemia in rat. Neurosci. Lett. 368, 87–91. Borutaite, V., Jekabsone, A., Morkuniene, R., Brown, G.C., 2003. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia. J. Mol. Cell. Cardiol. 35, 357–366.
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Chen, J.Q., Delannoy, M., Cooke, C., Yager, J.D., 2004. Mitochondrial localization of ERa and ERb in human MCF7 cells. Am. J. Physiol. 286, E1011–E1022. Datta, S.R., Dudek, H., Tao, X., Masters, S., Fu, H., Gotoh, Y., Greenberg, M.E., 1997. Akt phosphorylation of BAD couples survival signals to the cell-intrinsic death machinery. Cell 91, 231–241. Griffiths, E.J., Halestrap, A.P., 1995. Mitochondrial non-specific pores remain closed during cardiac ischaemia, but open upon reperfusion. Biochem. J. 307, 93–98. Hale, S.L., Birnbaum, Y., Cloner, R.A., 1996. Beta-estradiol, but not alfaestradiol, reduced myocardial necrosis in rabbits after ischemia and reperfusion. Am. Heart J. 132, 258–262. Ho, K.J., Liao, J.K., 2002. Non-nuclear actions of estrogen. Arterioscler. Thromb. Vasc. Biol. 22, 1952–1961. Jennings, R.B., 1984. Calcium ions in ischemia. In: Opie, L.H. (Ed.), Calcium Antagonists and Cardiovascular Disease. Raven Press, New York, pp. 85– 95. Liao, J.K., 2001. Cross-coupling between the oestrogen receptor and phosphoinositide 3-kinase. Biochem. Soc. Trans. 31, 66–70. Mendelsohn, M.E., Karas, R.H., 1999. The protective effects of estrogen on the cardiovascular system. N. Engl. J. Med. 340, 1801–1811. Moats, R.K., Ramirez, V.D., 1998. Rapid uptake and binding of estradiol17beta-6-(O-carboxymethyl)oxime:125I-labeled BSA by female rat liver. Biol. Reprod. 58, 531–538. Moats, R.K., Ramirez, V.D., 2000. Electron microscopic visualization of membrane-mediated uptake and translocation of estrogen-BSA: colloidal gold by hepG2 cells. J. Endocrinol. 166, 631–647.
Morkuniene, R., Jekabsone, A., Borutaite, V., 2002. Estrogens prevent calcium-induced release of cytochrome c from heart mitochondria. FEBS Lett. 521, 53–56. Nazareth, W., Yafei, N., Crompton, M., 1991. Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A. J. Mol. Cell. Cardiol. 23, 1351–1354. Patten, R.D., Pourati, I., Aronovitz, M.J., Baur, J., Celestin, F., Chen, X., Michael, A., Haq, S., Nuedling, S., Grohe, C., Force, T., Mendelsohn, M.E., Karas, R.H., 2004. 17Beta-estradiol reduces apoptosis in vivo and in vitro via activation of phospho-inositide-3kinase/Akt signaling. Circ. Res. 95, 692–699. Piper, H.M., Sezer, O., Schleyer, M., Schwartz, P., Hutter, J.F., Spieckermann, P.G.J., 1985. Development of ischemia-induced damage in defined mitochondrial subpopulations. Mol. Cell. Cardiol. 17, 885–896. Rieske, J.S., 1967. The quantitative determination of mitochondrial hemoproteins. Methods Enzymol. 10, 488–493. Sugishita, K., Li, F., Su, Z., Barry, W.H., 2003. Anti-oxidant effects of estrogens reduce [Ca2C]i during metabolic inhibition. J. Mol. Cell. Cardiol. 35, 331–336. Toleikis, A., Dzeja, P.P., Prashkyavicius, A.K., 1989. Functional state and the mechanisms of mitochondrial injury of ischemic heart. Sov. Med. Rev. A, Cardiol. 2, 95–132. Yang, S.H., Liu, R., Perez, E.J., Wen, Y., Stevent, S.M., Valencia, T., BrunZinkernagel, A.M., Prokai, L., Will, Y., Dykens, J., Koulen, P., Simpkins, J.W., 2004. Mitochondrial localization of estrogen receptor b. PNAS 101, 4130–4135. Zhai, P., Eurell, T.E., Cotthaus, R., Jeffery, E.H., Bahr, J.M., Gross, D.R., 2000. Effect of estrogen on global myocardial ischemia-reperfusion injury in female rats. Am. J. Physiol. 279, H2766–H2775.
Estradiol prevents release of cytochrome c from mitochondria and inhibits ischemia-induced apoptosis in perfused heart Ramune Morkuniene a,b, Odeta Arandarcikaite a, Vilmante Borutaite a,c,* a
Institute for Biomedical Research, Kaunas University of Medicine, Kaunas, Lithuania b Department of Biochemistry, Kaunas University of Medicine, Kaunas, Lithuania c Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK Received 28 October 2005; received in revised form 17 February 2006; accepted 21 February 2006 Available online 3 April 2006
Abstract The study investigated whether estradiol can prevent release of cytochrome c from mitochondria and induction of apoptosis after 30 and 60 min stop-flow heart ischemia in Langendorff-perfused female rat hearts. Pre-perfusion of hearts with 100 nM 17b-estradiol prevented the loss of cytochrome c from mitochondria, its accumulation in cytosol, and inhibition of respiration during ischemia. Estradiol strongly reduced activation of caspase-3-like activity and decreased DNA strand breaks in the nuclei of cardiomyocytes (measured by TUNEL staining). The results show that 17b-estradiol prevents the ischemia-induced release of cytochrome c from mitochondria, subsequent inhibition of mitochondrial respiration, and inhibits caspase activation and apoptosis. Therefore, inhibition of the intrinsic, mitochondria-mediated apoptotic pathway may be one of the mechanisms by which estrogens protect the heart against ischemic damage. q 2006 Elsevier Inc. All rights reserved. Keywords: Apoptosis; Estrogens; Ischemia; Mitochondria; Respiration; Cytochrome c
1. Introduction Estrogens are known to exert beneficial effects on the cardiovascular system after ischemia/reperfusion. It has been shown that 17b-estradiol reduces the extent of irreversible myocardial injury, ventricular arrhythmias, infarct size, and such effects have been detected in both male and female rats hearts (Hale et al., 1996; Zhai et al., 2000). A number of mechanisms have been proposed to explain the cardioprotective actions of estradiol in the heart. These include classical genomic or non-genomic responses. Estrogens act primarily through nuclear estrogen receptors (ERa and ERb), which regulate gene expression leading to the control of a variety of cellular functions (Mendelsohn and Karas, 1999). The second mechanism involves membrane or cytosolic estrogen receptors leading to transcriptional changes indirectly through activation of protein kinase cascades (Liao, 2001). However, recent evidence indicates that estrogens can also exert acute, * Corresponding author. Address: Department of Biochemistry, University of Cambridge, Tennis Court Road, Cambridge CB2 1QW, UK. Tel.: C44 1223 333342; fax: C44 1223 333345. E-mail address: [email protected] (V. Borutaite).
0531-5565/$ - see front matter q 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.exger.2006.02.010
transcription-independent effects on cellular functions (Babiker et al., 2002). These non-transcriptional actions of estrogens in myocardial ischemia/reperfusion have been suggested to be due to stimulation of NO production, inhibition of myocardial calcium accumulation or due to the antioxidant properties of estrogens (Zhai et al., 2000; Sugishita et al., 2003). We have previously shown that estrogens can acutely protect isolated heart mitochondria from the loss of cytochrome c induced by high calcium (Morkuniene et al., 2002), which is considered as one of the damaging factors during heart ischemia/reperfusion (Jennings, 1984). It was also known that cytochrome c release from mitochondria is one of the earliest mitochondrial events during ischemia (Piper et al., 1985; Toleikis et al., 1989), which causes inhibition of ATP synthesis, activation of caspases and nuclear apoptosis (Borutaite et al., 2003). In the present study, we aimed to elucidate whether estrogens can prevent activation of the intrinsic, mitochondriamediated apoptotic pathway in ischemic heart by protecting mitochondria from cytochrome c release. 2. Materials and methods Hearts from 2 months old, female Wistar rats were used in experiments. The hearts were perfused on Langendorff perfusion system with Krebs–Henseleit solution (11 mM
R. Morkuniene et al. / Experimental Gerontology 41 (2006) 704–708
glucose, 118 mM NaCl, 25 mM NaHCO3, 4.8 mM KCl, 1.2 mM KH2PO4, 1.2 mM CaCl2, 1.7 mM MgSO4 and 0.7 mM Na pyruvate, pH 7.2 at 37 8C). Hearts were washed for 5 min with Krebs–Henseleit solution, then 100 nM 17bestradiol was added and hearts were perfused for another 15 min. Control hearts were perfused for the same time but without estradiol. Coronary flow rate was 10–12 ml/min in control and estradiol-perfused hearts. After 15 min of perfusion with/without estradiol, stop-flow ischemia was induced for 30 or 60 min. Hearts were homogenized with a motor-driven teflon/glass homogenizer in the medium containing 180 mM KCl, 20 mM Tris–HCl, 1 mM EGTA, pH 7.3. Mitochondrial and cytosolic fractions were separated by differential centrifugation (5 min! 750g, 10 min!6800g, 30 min!10,000g). Mitochondrial respiration was measured with a Clarke-type oxygen electrode at 37 8C in 1 ml incubation buffer containing 110 mM KCl, 2.24 mM MgCl2, 10 mM Tris–HCl, 5 mM KH2PO4, 4 IU/ml creatine kinase, 50 mM creatine, 1 mM pyruvateC1 mM malate (pH 7.2) and 1 mM ATP as described in Borutaite et al. (2003). In some experiments mitochondrial respiration was measured in the presence of 30 mM exogenous cytochrome c. Total cytochrome c and cytochrome a content was determined in mitochondria solubilized with 1% Triton X-100 (w/v). Sodium hydrosulphite-reduced minus hydrogen-peroxide-oxidized absorption difference spectra were recorded with a Hitachi-557 spectrophotometer. Cytochrome c content was estimated by using the absorption difference at the wavelength pair 550/535 nm and 3Z14.5 mMK1 cmK1; for cytochrome a—wavelength pair 605/630 nm and 3Z 10.4 mMK1 cmK1 as described in Rieske (1967). Cytochrome c content in cytosolic fractions was detected using Quantikinine M rat/mouse Immunoassay ELISA kit (R&D Systems). Cytosolic fraction proteins were dissolved in 0.5% Triton X-100 and further procedures were performed according to manufacturer’s protocol. Activity of caspases was measured in cytosolic extracts using 0.1 mM z-DEVD-p-nitroanilide, a caspase-3 substrate, as described in Borutaite et al. (2003). The number of apoptotic cells was quantified by dUTP nick end labeling (TUNEL) using CardioTACS (R&D Systems) in situ apoptosis detection kit. Hearts were fixed in 10% formalin. The tissues were embedded in molten paraffin and frozen. All other procedures were performed according to the manufacturer’s protocol. The number of TUNEL-positive cardiomyocytes was counted in at least 10 different microscopic fields on each section containing approximately 150 cardiomyocytes in each. Data were expressed as meansGSE of 5–10 separate experiments and analyzed with ANOVA followed by Tukey’s multiple comparison test. 3. Results We investigated whether high physiological concentration of 17b-estradiol (100 nM) can prevent the release of
705
cytochrome c from mitochondria to cytosol during ischemia in perfused rat female hearts. As can be seen in Fig. 1A, content of cytochrome c in mitochondria decreased by 19 and 26% after 30 and 60 min of ischemia, respectively, compared to the control level. However, in mitochondria isolated from ischemic hearts pre-perfused with 100 nM estradiol the content of cytochrome c was not significantly different from control or controlCestradiol mitochondria (Fig. 1A). Ischemia or estradiol itself had no significant effect on the mitochondrial content of cytochrome a, an integral component of the inner membrane (data not shown) suggesting that there was no general degradation of mitochondrial proteins and the release was specific for cytochrome c. Sixty minutes ischemia-induced loss of cytochrome c from mitochondria was observed (though to a lesser extent) even when mitochondria were isolated in sucrose-based buffer known to reduce dissociation of cytochrome c from mitochondria with damaged outer membrane. And again, the loss of cytochrome c was completely prevented
Fig. 1. Estradiol prevents release of cytochrome c from heart mitochondria to cytosol during ischemia. (A) Mitochondrial content of cytochrome c. (B) Cytochrome c content in cytosolic fractions. Female rat hearts were perfused for 15 min with Krebs–Henseleit solution with or without 100 nM of 17bestradiol, then stop-flow ischemia was induced for 30 or 60 min. Content of cytochrome c in isolated mitochondria is expressed as nmol/mg of mitochondrial protein. Cytosolic cytochrome c content is expressed as nmol/mg of cytosolic protein. *, Statistically significant effect of ischemia (p!0.05) if compared to control; #, statistically significant effect of estradiol (p!0.05) if compared to treatment without estradiol in the same group (30 or 60 min ischemia).
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when hearts were treated with estradiol: cytochrome c levels were 0.572G0.022, 0.493G0.017 (p!0.05) and 0.568G 0.011 nmol/mg protein in control, 60 min ischemic and 60 min ischemic plus estradiol mitochondria. Concomitant with decrease of mitochondrial content of cytochrome c during ischemia there was accumulation of this protein in cytosols and this was prevented when hearts were perfused with estradiol before induction of ischemia (Fig. 1B). Cytochrome c content in cytosolic extracts prepared from 30 to 60 min ischemic hearts increased by 45 and 56%, respectively, compared to control, while in cytosolic extracts from ischemicCestradiol hearts there was no significant increase in cytochrome c concentration compared to control level. Some cytochrome c was always present in control cytosols with or without estradiol and this may be caused by some inevitable damage of mitochondria during homogenization procedure of heart tissue. Altogether these results show that estradiol can prevent ischemia-induced release of cytochrome c from mitochondria to the cytosol. Next we tested whether perfusion of the heart with estradiol can prevent ischemia-induced inhibition of mitochondrial respiration. As can be seen from Fig. 2A, mitochondrial state
3 respiration after 30 and 60 min ischemia was lower by 47 and 63% compared to control mitochondria oxidizing pyruvate and malate. However, in mitochondria from 30 to 60 min ischemic hearts pre-perfused with estradiol state 3 respiratory rate was higher by 23 and 31%, respectively, compared to the respiration of ischemia-damaged mitochondria in the absence of estradiol. Exogenous cytochrome c added to the mitochondrial incubation medium stimulated respiratory rate by 110% after 30 min ischemia and by 192% after 60 min ischemia compared with respiration of ischemia-damaged mitochondria without added cytochrome c (Fig. 2B). However, when hearts were perfused with estradiol, cytochrome c stimulated respiration only by 70 and 106% in mitochondria from 30 to 60 min ischemia, respectively. Exogenous cytochrome c stimulates respiration only if mitochondrial outer membrane is damaged and permeable to cytochrome c. Preparation of isolated mitochondria are always to some degree heterogenous in respect to intactness of mitochondrial outer membrane as part of mitochondria are inevitably damaged during isolation (Piper et al., 1985), and this may be the reason why added cytochrome c stimulated the respiration of control mitochondria. Thus the data suggest that inhibition of respiration after ischemia is partially due to the loss of cytochrome c from mitochondria, and that estradiol partially prevented ischemiainduced respiratory inhibition in part by protection of mitochondria from the loss of cytochrome c. It has been shown that prolonged heart ischemia causes activation of caspases and nuclear fragmentation (Borutaite et al., 2003). We investigated whether perfusion of the hearts with 17b-estradiol can prevent activation of caspases and subsequent nuclear apoptosis. Caspase-3-like, DEVD-cleaving activity was low in control, but increased significantly after 60 min of ischemia (Fig. 3A). Activity of caspases was significantly (though not completely) blocked when hearts were perfused with 17b-estradiol before induction of ischemia (Fig. 3A). To further characterize apoptosis in ischemic myocardium, we used the TUNEL method to determine cells with DNA strand breaks. As can be seen from Fig. 3B, cardiomyocytes with DNA strand breaks were practically not detectable in control hearts, however, the number of such cells increased 10 times during ischemia. Perfusion of hearts with estradiol substantially reduced the number of cardiomyocytes with DNA strand breaks after 60 min ischemia, which is consistent with reduced activity of caspases due to prevention of cytochrome c release from mitochondria after such treatment. 4. Discussion
Fig. 2. Estradiol protects mitochondrial respiration damaged by ischemia. (A) Respiration rate without exogenous cytochrome c. (B) Respiration rate in the presence of 30 mM exogenous cytochrome c. 1 mM pyruvateC1 mM malate were used as respiratory substrate. Other symbols are as in Fig. 1.
In the present study we have demonstrated that estradiol blocked ischemia-induced release of cytochrome c from heart mitochondria into cytosol, subsequent activation of caspases and apoptosis. Estradiol also prevented inhibition of respiration of ischemia-damaged heart mitochondria caused by loss of cytochrome c. Similarly, a recent study has reported that estradiol reduced cytochrome c translocation and minimized hippocampal damage caused by transient global ischemia in rat
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Fig. 3. Estradiol prevents ischemia-induced caspase activation and nuclear apoptosis. (A) Caspase-3-like, DEVD-cleaving activity in cytosolic fractions from control, 60 min ischemic or 60 min ischemic plus estradiol hearts; (B) DNA fragmentation in cardiomyocytes. Other symbols are as in Fig. 1. Total number of cardiomyocytes analyzed for DNA fragmentation varied from 4000 to 10,000 in each group.
brain (Bagetta et al., 2004). Therefore, part of the cardioprotective action of estradiol seems likely to be related to preventing mitochondrial loss of cytochrome c. The classical target of estrogens is the nucleus, however, recent studies indicate that exogenously added estrogens are also transported to mitochondria (Moats and Ramirez, 1998, 2000). In in vivo experiments on ovariectomized rats it has been shown that added estrogen translocated from plasma membrane to mitochondria rather than to nuclei in variety of tissues (Moats and Ramirez, 1998). Therefore, it is likely that perfusion of the hearts with estradiol may also lead to at least partial accumulation of this hormone in mitochondria where it can act on membranes directly or indirectly through estrogen receptors that were recently identified in mitochondria (Chen et al., 2004; Yang et al., 2004). In isolated mitochondria, exogenously added estradiol has been shown to inhibit mitochondrial permeability transition pore (PTP)-related release of cytochrome c from mitochondria induced by high calcium (Morkuniene et al., 2002). Elevation of intracellular calcium is known as a causative damaging factor during ischemia/reperfusion (Jennings, 1984), which besides other effects may induce opening of mitochondrial PTP. PTP
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opening at reperfusion after short times of myocardial ischemia has been documented (Griffiths and Halestrap, 1995), though some studies indicated PTP opening during prolonged ischemia alone (Borutaite et al., 2003; Nazareth et al., 1991). PTP can cause cytochrome c release from mitochondria due to swelling of the mitochondria and rupture of the outer membrane. Thus the protective effect of estradiol may be related to the inhibition of PTP. On the other hand, we cannot rule out the possibility that the protective effect of estradiol on mitochondria in the perfused heart may be indirect as several authors have shown that estradiol inhibits calcium accumulation in the cytosol during ischemia/reperfusion (Zhai et al., 2000), and this may reduce the probability of PTP pore opening during ischemia. Another potential mechanism of indirect action of estrogens on mitochondria preventing loss of cytochrome c may be related to the effect of the hormone on protein kinase cascades through its binding to estrogen receptor a. It has been demonstrated that estrogens can activate the phosphoinositol3 kinase (PI3K) and the protein kinase Akt (Liao, 2001; Ho and Liao, 2002; Patten et al., 2004), which may phosphorylate and inactivate the proapoptotic proteins Bad and Bax (Datta et al., 1997) known to induce release of cytochrome c from mitochondria leading to apoptotic cell death. Therefore, preservation of mitochondria from the loss of cytochrome c might be related to estradiol-ER-a complex induced protein kinase Akt activation resulting in modification of proapoptotic Bcl-2 family proteins. The possibility that estrogens inhibit Bax- or Bad-induced cytochrome c release from mitochondria needs to be investigated in further studies. In conclusion, the present study provides evidence that at high physiological concentrations estradiol protects heart mitochondria from ischemia-induced release of cytochrome c, subsequent inhibition of respiration, activation of caspases and DNA fragmentation, and this may be one of the mechanisms by which estrogens preserve myocardial cell viability during ischemia/reperfusion. Acknowledgements We thank Dr Guy Brown for critical reading of manuscript and fruitful discussion. This work was supported by Lithuanian State Science and Studies Foundation and the British Heart Foundation. References Babiker, F.A., De Windth, L.J., Eickels, M., Grohe, C., Meyer, R., Doevendans, P.A., 2002. Estrogenic hormone action in the heart: regulatory network and function. Cardiovasc. Res. 53, 709–719. Bagetta, G., Chiappetta, O., Amantea, D., Iannone, M., Rotiroti, D., Costa, A., Nappi, G., Corosaniti, M.T., 2004. Estradiol reduces cytochrome c translocation and minimizes hippocampal damage caused by transient global ischemia in rat. Neurosci. Lett. 368, 87–91. Borutaite, V., Jekabsone, A., Morkuniene, R., Brown, G.C., 2003. Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia. J. Mol. Cell. Cardiol. 35, 357–366.
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