, Volume 12, Issue 3, pp 465–474 | Cite as

Identification of an inhibitor of caspase activation from heart extracts; ATP blocks apoptosome formation

  • Afshin Samali
  • Martin O’Mahoney
  • Janice Reeve
  • Susan Logue
  • Eva Szegezdi
  • Jill McMahon
  • Howard O. FearnheadEmail author


By revealing the biochemistry of apoptosis it is expected we will both improve our understanding of diseases where apoptosis plays an important role and aid the development of therapies for these disorders. Caspases are a family of proteases whose activity is required for apoptosis. In this study, a cell-free system was used to investigate the mechanism of caspase-9 activation in extracts from heart cells. Unlike extracts from other cell types, heart extracts were found to activate caspases poorly. This could be explained by the low levels of Apaf-1 in heart cells. However, subsequent testing showed that heart extracts contained an inhibitor of caspase activation that could block caspase activation in extracts from different cell types. Subsequent purification of the inhibitor of caspase activation from these extracts identified ATP. Caspase-9 is activated by recruitment into a multi-protein complex, the apoptosome, which then activates downstream caspases that kill the cell. Importantly, size exclusion chromatography showed that ATP inhibits apoptosome formation at physiologically relevant concentrations. Together these data support the hypothesis that intracellular ATP concentration is a critical factor in determining whether an apoptotic stimulus can induce apoptosome formation. Thus, the well described fall in intracellular ATP apoptosis is not an epiphenomenon but may be a pro-apoptotic event contributing to cell death.


ATP Caspase Apoptosome Heart 



We would like to thank our colleagues at the NCBES, NUI Galway for advice and criticism. We would particularly like to acknowledge the invaluable contribution of Dr Dean Tang and Dhyan Chandra of the MD Anderson Cancer Center to timely completion this project and thank them for their openness and collegiality. JR is supported by is supported by an EMBARK scholarship from IRCSET. This research is funded by an HEA grant to AS and an HRB project grant (RP/2006/19) to HOF.


  1. 1.
    Kitsis RN, Mann DL (2005) Apoptosis and the heart: a decade of progress. J Mol Cell Cardiol 38:1–2PubMedCrossRefGoogle Scholar
  2. 2.
    Garg S, Narula J, Chandrashekhar Y (2005) Apoptosis and heart failure: clinical relevance and therapeutic target. J Mol Cell Cardiol 38:73–79PubMedCrossRefGoogle Scholar
  3. 3.
    Lowe SW, Cepero E, Evan G (2004) Intrinsic tumour suppression. Nature 432:307–315PubMedCrossRefGoogle Scholar
  4. 4.
    Salvesen GS (2002) Caspases and apoptosis. Essays Biochem 38:9–19PubMedGoogle Scholar
  5. 5.
    Boatright KM, Salvesen GS (2003) Caspase activation. Biochem Soc Symp 233–242Google Scholar
  6. 6.
    Alnemri ES (1997) Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 64:33–42PubMedCrossRefGoogle Scholar
  7. 7.
    Cecconi F (1999) Apaf1 and the apoptotic machinery. Cell Death Differ 6:1087–1098PubMedCrossRefGoogle Scholar
  8. 8.
    Kischkel FC, Hellbardt S, Behrmann I et al (1995) Cytotoxicity-dependent APO-1 (Fas/CD95)-associated proteins form a death-inducing signaling complex (DISC) with the receptor. Embo J 14:5579–5588PubMedGoogle Scholar
  9. 9.
    Slee EA, Harte MT, Kluck RM et al (1999) Ordering the cytochrome c-initiated caspase cascade: hierarchical activation of caspases-2, -3, -6, -7, -8, and -10 in a caspase-9-dependent manner. J Cell Biol 144:281–292PubMedCrossRefGoogle Scholar
  10. 10.
    Kajstura J, Cheng W, Reiss K et al (1996) Apoptotic and necrotic myocyte cell deaths are independent contributing variables of infarct size in rats. Lab Invest 74:86–107PubMedGoogle Scholar
  11. 11.
    Yue TL, Ma XL, Wang X et al (1998) Possible involvement of stress-activated protein kinase signaling pathway and Fas receptor expression in prevention of ischemia/reperfusion-induced cardiomyocyte apoptosis by carvedilol. Circ Res 82:166–174PubMedGoogle Scholar
  12. 12.
    Potts MB, Vaughn AE, McDonough H, Patterson C, Deshmukh M (2005) Reduced Apaf-1 levels in cardiomyocytes engage strict regulation of apoptosis by endogenous XIAP. J Cell Biol 171:925–930PubMedCrossRefGoogle Scholar
  13. 13.
    Bialik S, Cryns VL, Drincic A et al (1999) The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes. Circ Res 85:403–414PubMedGoogle Scholar
  14. 14.
    Sanchis D, Mayorga M, Ballester M, Comella JX (2003) Lack of Apaf-1 expression confers resistance to cytochrome c-driven apoptosis in cardiomyocytes. Cell Death Differ 10:977–986PubMedCrossRefGoogle Scholar
  15. 15.
    Szegezdi E, Duffy A, O’Mahoney ME et al (2006) ER stress contributes to ischemia-induced cardiomyocyte apoptosis. Biochem Biophys Res CommunGoogle Scholar
  16. 16.
    Nakagawa T, Zhu H, Morishima N et al (2000) Caspase-12 mediates endoplasmic-reticulum-specific apoptosis and cytotoxicity by amyloid-beta. Nature 403:98–103PubMedCrossRefGoogle Scholar
  17. 17.
    Hitomi J, Katayama T, Taniguchi M et al (2004) Apoptosis induced by endoplasmic reticulum stress depends on activation of caspase-3 via caspase-12. Neurosci Lett 357:127–130PubMedCrossRefGoogle Scholar
  18. 18.
    Hitomi J, Katayama T, Eguchi Y et al (2004) Involvement of caspase-4 in endoplasmic reticulum stress-induced apoptosis and Abeta-induced cell death. J Cell Biol 165:347–356PubMedCrossRefGoogle Scholar
  19. 19.
    Genini D, Budihardjo I, Plunkett W et al (2000) Nucleotide requirements for the in vitro activation of the apoptosis protein-activating factor-1-mediated caspase pathway. J Biol Chem 275:29–34PubMedCrossRefGoogle Scholar
  20. 20.
    Liu X, Kim CN, Yang J, Jemmerson R, Wang X (1996) Induction of apoptotic program in cell-free extracts: requirement for dATP and cytochrome c. Cell 86:147–157PubMedCrossRefGoogle Scholar
  21. 21.
    Zou H, Henzel WJ, Liu X, Lutschg A, Wang X (1997) Apaf-1, a human protein homologous to C. elegans CED-4, participates in cytochrome c-dependent activation of caspase-3. Cell 90:405–413PubMedCrossRefGoogle Scholar
  22. 22.
    Li P, Nijhawan D, Budihardjo I et al (1997) Cytochrome c and dATP-dependent formation of Apaf-1/caspase-9 complex initiates an apoptotic protease cascade. Cell 91:479–489PubMedCrossRefGoogle Scholar
  23. 23.
    O’Mahoney ME, Logue S, Szegezdi E, Stenson-Cox C, Fitzgerald U, Samali A (2003) Hypoxia and ischemia induce nuclear condensation and caspase activation in cardiomyocytes. Ann N Y Acad Sci 1010:728–732PubMedCrossRefGoogle Scholar
  24. 24.
    Chereau D, Zou H, Spada AP, Wu JC (2005) A nucleotide binding site in caspase-9 regulates apoptosome activation. Biochemistry 44:4971–4976PubMedCrossRefGoogle Scholar
  25. 25.
    Chandra D, Bratton SB, Person MD et al (2006) Intracellular nucleotides act as critical prosurvival factors by binding to cytochrome c and inhibiting apoptosome. Cell 125:1333–1346PubMedCrossRefGoogle Scholar
  26. 26.
    Izyumov DS, Avetisyan AV, Pletjushkina OY et al (2004) “Wages of fear”: transient threefold decrease in intracellular ATP level imposes apoptosis. Biochim Biophys Acta 1658:141–147PubMedCrossRefGoogle Scholar
  27. 27.
    Feldenberg LR, Thevananther S, del Rio M, de Leon M, Devarajan P (1999) Partial ATP depletion induces Fas- and caspase-mediated apoptosis in MDCK cells. Am J Physiol 276:F837–846PubMedGoogle Scholar
  28. 28.
    Jeyaseelan R, Poizat C, Wu HY, Kedes L (1997) Molecular mechanisms of doxorubicin-induced cardiomyopathy. Selective suppression of Reiske iron-sulfur protein, ADP/ATP translocase, and phosphofructokinase genes is associated with ATP depletion in rat cardiomyocytes. J Biol Chem 272:5828–5832PubMedCrossRefGoogle Scholar
  29. 29.
    Dang CV, Semenza GL (1999) Oncogenic alterations of metabolism. Trends Biochem Sci 24:68–72PubMedCrossRefGoogle Scholar
  30. 30.
    Belzacq AS, Vieira HL, Verrier F et al (2003) Bcl-2 and Bax modulate adenine nucleotide translocase activity. Cancer Res 63:541–546PubMedGoogle Scholar
  31. 31.
    Hardie DG, Hawley SA (2001) AMP-activated protein kinase: the energy charge hypothesis revisited. Bioessays 23:1112–1119PubMedCrossRefGoogle Scholar
  32. 32.
    Meisse D, Van de Casteele M, Beauloye C et al (2002) Sustained activation of AMP-activated protein kinase induces c-Jun N-terminal kinase activation and apoptosis in liver cells. FEBS Lett 526:38–42PubMedCrossRefGoogle Scholar
  33. 33.
    Capano M, Crompton M (2006) Bax translocates to mitochondria of heart cells during simulated ischaemia: involvement of AMP-activated and p38 mitogen-activated protein kinases. Biochem J 395:57–64PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2007

Authors and Affiliations

  • Afshin Samali
    • 1
  • Martin O’Mahoney
    • 1
  • Janice Reeve
    • 1
  • Susan Logue
    • 1
  • Eva Szegezdi
    • 1
  • Jill McMahon
    • 2
  • Howard O. Fearnhead
    • 2
    Email author
  1. 1.Cell Stress and Apoptosis Laboratory, National Centre Biomedical Engineering ScienceNational University of IrelandGalwayIreland
  2. 2.Caspase Laboratory, National Centre Biomedical Engineering ScienceNational University of IrelandGalwayIreland

Personalised recommendations