Original Article
Inhibition of mitochondrial permeability transition prevents mitochondrial dysfunction, cytochrome c release and apoptosis induced by heart ischemia

https://doi.org/10.1016/S0022-2828(03)00005-1Get rights and content

Abstract

Ischemia/reperfusion of heart causes contractile dysfunction, necrosis and/or apoptosis and is a major cause of human death, but the molecular mechanisms are unclear. We show that ischemia alone (without reperfusion) is sufficient to induce apoptosis and mitochondrial dysfunction, and we have investigated the mechanism responsible; 30 and 60Ā min stop-flow ischemia in Langendorff-perfused rat hearts induced progressive (a) release of cytochrome c from mitochondria to cytosol, (b) inhibition of the mitochondrial respiratory functions, (c) activation of caspase-3-like protease activity and (d) DNA strand breaks (however, only 2% of myocyte nuclei were TUNEL positive at 60Ā min). Fifteen minutes pre-perfusion of hearts with cyclosporin A, an inhibitor of mitochondrial-permeability transition (MPT), largely prevented all these ischemic changes. Pre-perfusion of hearts with FK506, an inhibitor of calcineurin, caused no protection. Pre-perfusion with DEVD-CHO, an inhibitor of caspase-3-like proteases, completely prevented ischemia-induced DNA strand breaks, but only partially blocked cytochrome c release and mitochondrial respiratory inhibition. Reperfusion of hearts after 30Ā min ischemia further stimulated caspase activity and nuclear apoptosis. We conclude that ischemia-induced MPT causes release of cytochrome c, which then activates the caspases that execute apoptosis and feedback to cause further cytochrome c release. The MPT-induced cytochrome c release is also largely responsible for the ischemic respiratory inhibition, which might contribute to contractile dysfunction or necrosis at reperfusion.

Introduction

Recent evidence indicates that ischemia/reperfusion can induce apoptosis in heart myocytes both in animals and humans [1], [2], [3], [4], as indicated by DNA laddering, DNA fragmentation and/or caspase activation. However, the mechanism by which apoptosis is induced by ischemia and/or reperfusion is still unclear. Apoptosis is executed by a family of cysteine proteases-caspases, which can be activated by two main pathways (see Ref. [5] for review). The first pathway is initiated by binding of extracellular ligands (Fas ligand, TNF-Ī±) to their cell-surface receptors leading to caspase-8 activation. The second is mediated by mitochondria, which release apoptotogenic proteins, mainly cytochrome c. Cytochrome c once released into the cytosol binds to a cytosolic protein Apaf-1, and this complex in the presence of dATP or ATP facilitates activation of caspase-9, which in turn activates downstream caspase-3. While these pathways have been defined in cultured cells undergoing apoptosis induced by various stimuli, little is known as to which caspase-activation pathway is triggered in the ischemic/reperfused heart. We and others have shown that ischemia/reperfusion of heart induces both cytochrome c release and caspase activation [2], [6], [7], but it is unclear whether cytochrome c release causes caspase activation or caspase activation causes cytochrome c release.

At least two ways of inducing cytochrome c release from mitochondria have been suggested by translocation from cytosol to mitochondria of BH3-domain-containing pro-apoptotic proteins (such as Bid, Bax or Bad) [8], [9], [10], or by mitochondrial-permeability transition (MPT) [11], [12]. MPT can be triggered in response to stress signals, such as high Ca2+ or reactive oxygen species [13], [14], [15], [16], conditions that are likely during ischemia/reperfusion. It is thought that MPT results from opening of a pore (the MPT pore) in mitochondrial membranes, which is formed by several proteins (including, probably, the voltage-dependent anion channel, ATP/ADP-translocator, cyclophilin D and creatine kinase) [13]. Upon MPT pore opening, the mitochondrial membrane potential collapses and proteins from the intermembrane space (including cytochrome c) can be released [11], [17]. There is evidence that the MPT pore may open during reperfusion [15], [18], [19]; however, it is unclear whether this may occur during ischemia itself. In addition to apoptosis, heart ischemia/reperfusion induces myocyte necrosis and contractile dysfunction after reperfusion. The mechanisms by which necrosis is induced are also unclear, but have been suggested to include mitochondrial respiratory inhibition, cellular ATP depletion, Ca2+ influx, ROS production, MPT and hypercontracture [20], [21]. Irreversible respiratory inhibition has partly been attributed to cytochrome c release [22], and may contribute to necrosis and contractile dysfunction after reperfusion.

In the research reported here, we show that ischemia alone (without reperfusion) can induce cytochrome c release, mitochondrial dysfunction, caspase activation and nuclear apoptosis, and that all these ischemic events can be prevented by blocking the MPT pore.

Section snippets

Methods

Hearts from male Wistar rats were used in the experiments. Hearts were perfused using a Langendorff-perfusion system with Krebsā€“Henseleit solution (11Ā mM glucose, 118Ā mM NaCl, 25Ā mM NaHCO3, 4.8Ā mM KCl, 1.2Ā mM KH2PO4, 1.2Ā mM CaCl2, 1.7Ā mM MgSO4 and 0.7Ā mM Na pyruvate; pH 7.2 at 37Ā Ā°C), with or without 0.1Ā Ī¼M cyclosporin A (Sigma), 25Ā Ī¼M FK506 (Calbiochem) or 1Ā Ī¼M DEVD-CHO (cell permeable caspase-3 inhibitor with N-terminal sequence, which facilitates the peptide's cell permeability; Biomol).

Cyclosporin A prevents loss of cytochrome c from mitochondria during myocardial ischemia

We have previously shown that ischemia causes the release of mitochondrial cytochrome c in isolated rat heart [7], [22]. To test whether this cytochrome c release is due to MPT, we pre-perfused hearts with a MPT inhibitor cyclosporin A (0.1Ā Ī¼M), then induced ischemia and measured the cytochrome c content of subsequently isolated mitochondria. Fig.Ā 1 shows the effects of ischemia and cyclosporin A on mitochondrial content of cytochromes c and a. After 30 and 60Ā min ischemia, the mitochondrial

Discussion

Ischemia alone (in the absence of reperfusion) caused substantial cytochrome c release from the mitochondria to the cytosol, without gross damage to the mitochondria. This cytochrome c release was largely prevented by pre-perfusion of the hearts with 0.1Ā Ī¼M cyclosporin A, a concentration known to block ischemia-induced MPT in heart [18], [19]. Care must be taken when interpreting results with cyclosporin A as it can also inhibit calcineurin (Ca2+/calmodulin-dependent protein phospatase).

Acknowledgements

The work was supported by British Heart Foundation and FEBS fellowship to A.J.

References (43)

  • E.J. Griffiths et al.

    Protection by cyclosporin A of ischemia/reperfusion-induced damage in isolated rat hearts

    J Mol Cell Cardiol

    (1993)
  • W. Nazareth et al.

    Inhibition of anoxia-induced injury in heart myocytes by cyclosporin A

    J Mol Cell Cardiol

    (1991)
  • M. Chen et al.

    Calpain and mitochondria in ischemia/reperfusion injury

    J Biol Chem

    (2002)
  • B. Freude et al.

    Apoptosis is initiated by myocardial ischemia and executed during reperfusion

    J Mol Cell Cardiol

    (2000)
  • S.C. Black et al.

    Co-localization of the cysteine protease caspase-3 with apoptotic myocytes after in vivo myocardial ischemia and reperfusion in the rat

    J Mol Cell Cardiol

    (1998)
  • R.A. Gottlieb et al.

    Reperfusion injury induces apoptosis in rabbit cardiomyocytes

    J Clin Invest

    (1994)
  • S. Bialik et al.

    The mitochondrial apoptotic pathway is activated by serum and glucose deprivation in cardiac myocytes

    Circ Res

    (1999)
  • G. Olivetti et al.

    Apoptosis in the failing human heart

    N Engl J M

    (1997)
  • A. Saraste et al.

    Apoptosis in human acute myocardial infarction

    Circulation

    (1997)
  • I. Budihardjo et al.

    Biochemical pathways of caspase activation during apoptosis

    Annu Rev Cell Dev Biol

    (1999)
  • D. De Moissac et al.

    Caspase activation and mitochondrial cytochrome c release during hypoxia-mediated apoptosis of adult ventricular myocytes

    J Mol Cell Cardiol

    (2000)
  • Cited by (171)

    View all citing articles on Scopus
    View full text