Apoptosis Repressor with Caspase Recruitment Domain Protects against Cell Death by Interfering with Bax Activation

Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B. , Sayen, M. R. , Williams, S. D. , Crow, M. T. , and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors. Myocardial ischemia/reperfusion (I/R) is associated with an extensive loss of myocardial cells. The apoptosis repressor with caspase recruitment domain (ARC) is a protein that is highly expressed in heart and skeletal muscle and has been demonstrated to protect the heart against I/R injury (Gustafsson, A. B. , Sayen, M. R. , Williams, S. D. , Crow, M. T. , and Gottlieb, R. A. (2002) Circulation 106, 735-739). In this study, we have shown that transduction of TAT-ARCL31F, a mutant of ARC in the caspase recruitment domain, did not reduce creatine kinase release and infarct size after I/R. TAT-ARCL31F also failed to protect against hydrogen peroxide-mediated cell death in H9c2 cells, suggesting that the caspase recruitment domain is important in mediating ARC's protective effects. In addition, we report that ARC co-immunoprecipitated with the pro-apoptotic protein Bax, which causes cytochrome c release when activated. TAT-ARC, but not TAT-ARCL31F, prevented Bax activation and cytochrome c release in hydrogen peroxide-treated H9c2 cells. TAT-ARC was also effective in blocking cytochrome c release after ischemia and reperfusion, whereas TAT-ARCL31F had no effect on cytochrome c release. In addition, recombinant ARC protein abrogated Bax-induced cytochrome c release from isolated mitochondria. This suggests that ARC can protect against cell death by interfering with activation of the mitochondrial death pathway through the interaction with Bax, preventing mitochondrial dysfunction and release of pro-apoptotic factors. Apoptosis plays an important role in the cardiovascular system in the process of homeostasis and development but also plays a role in the pathogenesis of certain diseases. Apoptosis is implicated in the death of myocytes in animal models of myocardial ischemia (2Gottlieb R. A. Burleson K. O. Kloner R. A. Babior B. M. Engler R. L. J. Clin. Investig. 1994; 94: 1621-1628Crossref PubMed Scopus (1357) Google Scholar), in humans with acute myocardial infarction (3Saraste A. Pulkki K. Kallajoki M. Henriksen K. Parvinen M. Voipio-Pulkki L. Circulation. 1997; 95: 320-323Crossref PubMed Scopus (751) Google Scholar, 4Olivetti G. Abbi R. Quaini F. Kajstura J. Cheng W. Nitahara J. A. Quaini E. Di Loreto C. Beltrami C. A. Krajewski S. Reed J. C. Anversa P. N. Engl. J. Med. 1997; 336: 1131-1141Crossref PubMed Scopus (1485) Google Scholar), and in congestive heart failure (5Narula J. Haider N. Virmani R. DiSalvo T. G. Kolodgie F. D. Hajjar R. J. Schmidt U. Semigran M. J. Dec G. W. Khaw B. -A. N. Engl. J. Med. 1996; 335: 1182-1189Crossref PubMed Scopus (1252) Google Scholar). Even though it is clear that the extensive death of cardiac myocytes after I/R 1The abbreviations used are: I/R, ischemia/reperfusion; CARD, caspase recruitment domain; ARC, apoptosis repressor with CARD; PBS, phosphate-buffered saline; MOPS, 4-morpholinepropanesulfonic acid. contributes to the decline of ventricular function and mortality, the pathways that initiate apoptosis during I/R in the heart are poorly understood. Studies have suggested that many factors, including Bcl-2 homologous proteins, ATP depletion, acidosis, calcium fluxes, and reactive oxygen species, cause cytochrome c release and apoptosis during I/R in cardiac myocytes (6Misao J. Hayakawa Y. Ohno M. Kato S. 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Apoptosis repressor with CARD (ARC) is a ∼22 kDa protein expressed at high levels in heart and skeletal muscle (13Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (311) Google Scholar). ARC was initially reported to interact with caspase-2 and -8 and to inhibit apoptosis induced by caspase-8 and receptor-induced apoptosis by Fas and TNF-R1, leading to the proposal that ARC protects by interfering with the function of initiator caspases (13Koseki T. Inohara N. Chen S. Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (311) Google Scholar, 14Li P. F. Li J. Muller E. C. Otto A. Dietz R. von Harsdorf R. Mol. Cell. 2002; 10: 247-258Abstract Full Text Full Text PDF PubMed Scopus (142) Google Scholar). However, other studies have reported that ARC-mediated protection may not necessarily operate through the inhibition of caspases. For instance, ARC suppressed hypoxia-induced apoptosis by inhibiting cytochrome c release from the mitochondria in a caspase-independent manner (15Ekhterae D. Lin Z. Lundberg M. S. Crow M. T. Brosius F. C. Nunez G. Circ. Res. 1999; 85: E70-E77Crossref PubMed Google Scholar). In addition, ARC overexpression inhibited oxidant stress-induced cell death in H9c2 cells by preserving mitochondrial function independently of caspase inhibition, suggesting that ARC might act at the level of the mitochondria (16Neuss M. Monticone R. Lundberg M. S. Chesley A. T. Fleck E. Crow M. T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). We recently reported that ARC can protect against I/R injury, where perfusion of TAT-ARC into isolated rat hearts reduced both creatine kinase release and infarct size (1Gustafsson A. B. Sayen M. R. Williams S. D. Crow M. T. Gottlieb R. A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar). Thus, we hypothesized that ARC might interfere with the activation of the mitochondrial apoptotic pathway in ischemia. Apoptosis that occurs through the mitochondrial pathway is partly regulated by Bcl-2 family proteins. Expression of Bcl-2 family proteins has been described in both developing and adult cardiac myocytes (6Misao J. Hayakawa Y. Ohno M. Kato S. Fujiwara T. Fujiwara H. Circulation. 1996; 94: 1506-1512Crossref PubMed Scopus (327) Google Scholar, 17Cook S. A. Sugden P. H. Clerk A. Circ. Res. 1999; 85: 940-949Crossref PubMed Scopus (231) Google Scholar, 18Kang P. M. Haunstetter A. Aoki H. Usheva A. Izumo S. Circ. Res. 2000; 87: 118-125Crossref PubMed Scopus (298) Google Scholar, 19Kajstura J. Cheng W. Reiss K. Clark W. A. Sonnenblick E. H. Krajewski S. Reed J. C. Olivetti G. Anversa P. Lab. Investig. 1996; 74: 86-107PubMed Google Scholar, 20Condorelli G. Morisco C. Stassi G. Notte A. Farina F. Sgaramella G. de Rienzo A. Roncarati R. Trimarco B. Lembo G. 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In this study, we have examined the role of ARC and ARCL31F in I/R injury and hydrogen peroxide-induced cell death and their effect on Bax. We demonstrate that the CARD domain is important in mediating the protective effects of ARC. In addition, we report that ARC interacts with Bax, which prevents its activation and subsequent cytochrome c release from the mitochondria. Recombinant Protein Expression and Purification—Recombinant TAT-fusion proteins were expressed and purified under denaturing conditions as described previously (1Gustafsson A. B. Sayen M. R. Williams S. D. Crow M. T. Gottlieb R. A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar). His-tagged ARC and ARCL31F were purified under native conditions. In brief, cells from 1-liter cultures were resuspended in native buffer (50 mm NaH2PO4, pH 8. 0, and 300 mm NaCl), followed by sonication. After centrifugation at 20, 000 × g for 20 min, the supernatants were added to columns containing nickel-nitriloacetic acid (Qiagen). The resin was washed with native buffer plus 20 mm imidazole and the proteins were eluted with 250 mm imidazole in the same buffer, followed by de-salting on PD-10 columns (Amersham Bisociences). GST-Bax (1-171) was grown in BL21 (DE) cells (Invitrogen) and expression was induced with 0. 75 mm isopropyl β-d-thiogalactoside for 4 h. The bacteria were recovered by centrifugation, and the pellet was resuspended in PBS and protease inhibitors and sonicated on ice. Triton X-100 was added to a final concentration of 1% and incubated for 30 min on a rocker at 4 °C. After centrifugation at 20, 000 × g for 15 min at 4 °C, the supernatant was mixed with glutathione-Agarose (Sigma) for 30 min at room temperature. The beads were then washed three times with PBS, and the protein was eluted with 10 mm reduced glutathione. Langendorff Perfusion and Ischemia/Reperfusion—Male Sprague-Dawley rats (225-250g) were anesthetized, and hearts were rapidly excised and cannulated onto the Langendorff perfusion apparatus using a protocol adapted from Tsuchida et al. (24Tsuchida A. Liu Y. Liu G. S. Cohen M. V. Downey J. M. Circ. Res. 1994; 75: 576-585Crossref PubMed Scopus (267) Google Scholar). The hearts were perfused with Krebs-Ringer buffer (with or without 50 nm TAT protein) at a constant pressure of 60 mm Hg. Perfused hearts were stabilized for 20 min before being subjected to 30 min of global ischemia and up to 120 min of reperfusion. The creatine kinase activity in the coronary effluent was measured using a diagnostic kit (Sigma) and infarct size was measured using triphenyl tetrazolium staining (1Gustafsson A. B. Sayen M. R. Williams S. D. Crow M. T. Gottlieb R. A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar). All animal procedures were approved by the Animal Care and Use Committee of The Scripps Research Institute. Preparation of Mitochondria and Cytosol—At the end of perfusion, the ventricles were minced and briefly homogenized with the use of a Polytron homogenizer (Kinematica, Basel, Switzerland) in ice-cold buffer A (220 mm mannitol, 70 mm sucrose, 2 mm EGTA, 5 mm MOPS, pH 7. 4, and 0. 2% bovine serum albumin). The homogenates were centrifuged for 10 min at 600 × g to remove unbroken tissue and nuclei, and the supernatants were centrifuged for 10 min at 3, 000 × g to pellet mitochondria. The supernatant was further centrifuged for 30 min at 100, 000 × g to obtain cytosol. The mitochondrial pellet was washed twice in buffer A and resuspended in incubation buffer containing 180 mm mannitol, 70 mm sucrose, 10 mm KCl, 10 mm MgCl2, 1 mm EGTA, 5 mm KH2PO4, and 10 mm MOPS, pH 7. 4. Immunoprecipitation and Western Blot Analysis—Rat hearts were homogenized using a Polytron homogenizer in lysis buffer containing 50 mm Tris-HCl, pH 7. 4, 150 mm NaCl, 1 mm EGTA, 1 mm EDTA, 1% Triton X-100, and complete protease inhibitor mixture (Roche). Lysates were incubated on ice for 30 min and then cleared by centrifugation at 20, 000 × g for 20 min. The supernatants were incubated with or at 4 °C. The of protein or protein were added to the and incubated on rocker for 2 at 4 °C. The beads were washed times in PBS and resuspended in of were on to and with or The protein were by the with bovine serum H9c2 cells were grown on for before transduction with proteins. TAT-fusion proteins were added to the cells for 1 followed by with hydrogen For of mitochondrial and cells were with 1 for 30 min at the end of the with and with 30 for the 5 min, followed by of cells with of 10 cells were and using was measured as the of of to the of in was measured as the of with by the of in For Bax H9c2 cells were in after with hydrogen with Triton X-100 in PBS, and then in serum in The cells were incubated for 1 with and in blocking washed three times in PBS, and then incubated with or to or After a the cells were washed three times with in the 30 of was added to the cells. All were with a and with a c cells on were with nm of TAT-ARC or TAT-ARCL31F for 1 in for 1 and then with 600 or for 4 h. the end of the cells were by washed with PBS, and resuspended in of buffer mm sucrose, 1 mm EGTA, pH 7. 4, 20 mm MOPS, pH 7. 4, 10 mm and The cells were by at for 5 min, and was added to the cells to a final concentration of 300 cells and were by centrifugation at × g for 5 min. The supernatants were centrifuged at × g for 10 min to pellet mitochondria. The were resuspended in buffer mm sucrose, 1 mm EGTA, and 20 mm MOPS, pH washed and resuspended in of buffer C. of mitochondrial protein were onto (Invitrogen) and to for Western c was by c and by For cytochrome c release mitochondria were isolated from rat hearts as described recombinant Bax was with or without recombinant ARC or ARCL31F for 10 min at 4 °C. The were then mixed with of mitochondria at 30 for in a final of 60 Mitochondria were by centrifugation at × g for 10 min. The and supernatants were by for cytochrome For in Bax of recombinant Bax was with 1 ARC or ARCL31F in After a incubation at 30 °C, of lysis buffer was added to were at 4 in the of 5 of Protein was added to incubated for 2 recovered by centrifugation, and washed times in ice-cold to the beads were eluted by of 50 of buffer and for 5 min. proteins were by and Western of TAT-ARC and TAT-ARCL31F on Myocardial CARD is a that is in many proteins in the of cell death pathways and is to through interaction K. P. J. Biochem. Sci. 1997; Full Text PDF PubMed Scopus Google Scholar). the CARD is important in mediating the protective effects of ARC in I/R injury, we perfused hearts with TAT-ARC or TAT-ARCL31F, a mutant of ARC with a acid in the of the of this in other proteins has previously been shown to to a of and protein H. 1997; PubMed Scopus Google Scholar, J. J. H. H. G. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus (281) Google Scholar). reported previously (1Gustafsson A. B. Sayen M. R. Williams S. D. Crow M. T. Gottlieb R. A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google and as shown in perfusion with TAT-ARC reduced both infarct size and creatine kinase release after I/R. In perfusion of hearts with TAT-ARCL31F did not reduce creatine kinase release or infarct size after I/R, suggesting that the CARD domain is important for ARC function in mediating a protective effect in the of TAT-ARC and TAT-ARCL31F on has been shown to inhibit hydrogen peroxide-induced cell death in H9c2 cells (1Gustafsson A. B. Sayen M. R. Williams S. D. Crow M. T. Gottlieb R. A. Circulation. 2002; 106: 735-739Crossref PubMed Scopus (116) Google Scholar, M. Monticone R. Lundberg M. S. Chesley A. T. Fleck E. Crow M. T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). the CARD is important in against we TAT-ARCL31F protect against cell 2 that of H9c2 cells with hydrogen loss of mitochondrial and and that TAT-ARCL31F did not protect H9c2 cells against hydrogen peroxide-mediated cell death in the the that it with the of ARC. demonstrate that the CARD domain of ARC is important in against of ARC with are the of of pro-apoptotic Bcl-2 proteins that to by causing the release of factors, including cytochrome from the mitochondria. overexpression of ARC can protect the mitochondria and cytochrome c release in response to and (15Ekhterae D. Lin Z. Lundberg M. S. Crow M. T. Brosius F. C. Nunez G. Circ. Res. 1999; 85: E70-E77Crossref PubMed Google Scholar, M. Monticone R. Lundberg M. S. Chesley A. T. Fleck E. Crow M. T. J. Biol. Chem. 2001; 276: 33915-33922Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar), we hypothesized that ARC might have an effect on the pro-apoptotic protein Bax in the ARC with Bax, we examined the interaction ARC and Bax in the heart by of Bax from rat heart and then of the by for of ARC. In a ARC was for of Bax. shown in ARC was in the by using and Bax was in the In addition, to Bax with TAT-ARC, we perfused rat hearts with TAT-ARC, or TAT-ARCL31F, followed by and of Bax. of Bax with an that both TAT-ARC and TAT-ARCL31F, but not co-immunoprecipitated with Bax that a interaction ARC and Bax and that this interaction might interfere with Bax of ARC on Bax and c Bax activation can inhibited by ARC, we H9c2 cells with TAT-ARC or TAT-ARCL31F followed by 2 of hydrogen We activation of Bax using an that the of Bax S. A. A. R. Montessuit S. S. Maundrell K. B. Martinou J. C. J. 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We cytochrome c release in H9c2 cells with hydrogen and the of ARC or ARCL31F inhibit hydrogen peroxide-induced cytochrome c release. in the loss of cytochrome c from the mitochondria in H9c2 cells, whereas cells with TAT-ARC did not release cytochrome c from the mitochondria after hydrogen was to cytochrome c release in the of we that the of cytochrome c in the in rat hearts subjected to I/R perfused with TAT-ARC, but not TAT-ARCL31F, reduced cytochrome c release after I/R. that ARC is blocking cytochrome c release by inhibiting Bax activation in the ARC inhibit Bax-induced cytochrome c we the of ARC to inhibit cytochrome c release from mitochondria isolated from the rat of recombinant Bax to mitochondria in in the release of cytochrome c J. M. Xie Z. Deveraux Q. Ellerby L. Bredesen D. Reed J. C. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 4997-5002Crossref PubMed Scopus (1375) Google Scholar). The in that Bax induced cytochrome c release from isolated heart mitochondria. of Bax with recombinant purified ARC reduced cytochrome c release from isolated that the effect of ARC in these mitochondria is to interfere with Bax function In ARCL31F did not interfere with the effects of Bax on isolated mitochondria. We the of ARC and ARCL31F to to recombinant Bax. with the with the proteins from and heart we that both of ARC to Bax in a system of the recombinant proteins that the interaction ARC and Bax not the CARD domain, but the of ARC to Bax function is CARD In addition, it that or proteins are not for the ARC is expressed at high levels in the with the to apoptosis in the We have previously shown that the levels of ARC through protein transduction can reduce the of cell death after myocardial ischemia and reperfusion. In the study, we have to the for We that the CARD domain of ARC is for its protective of to at is to CARD with in other proteins H. 1997; PubMed Scopus Google Scholar, J. J. H. H. G. Cell. 1998; 94: Full Text Full Text PDF PubMed Scopus (281) Google Scholar). of mitochondrial is for and preventing with ARC was to mitochondrial and cytochrome c release in H9c2 cells to hydrogen and in the isolated perfused heart subjected to ischemia and reperfusion. Bax has been shown to a of the mitochondrial pathway of hydrogen to the of we that Bax the of activation in H9c2 cells. ARC interacts with Bax, as demonstrated by of ARC and Bax. We that a as or that an containing ARC and Bax H. Q. Krajewski S. M. Xie Z. S. S. A. Reed J. C. Proc. Natl. Acad. Sci. U. S. A. 2000; PubMed Scopus Google Scholar, Cheng E. H. J. M. Mol. Cell. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). 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Nunez G. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 5156-5160Crossref PubMed Scopus (311) Google and through D. S. J. PubMed Scopus Google Scholar). ARC has also been shown to by preserving mitochondrial J. A. D. V. L. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). these that ARC prevents cell death by interfering with We N. Kitsis for