Inhibition of HtrA2/Omi ameliorates heart dysfunction following ischemia/reperfusion injury in rat heart in vivo

https://doi.org/10.1016/j.ejphar.2006.10.067Get rights and content

Abstract

High temperature requirement A2 (HtrA2)/Omi is a mitochondrial serine protease that is released into the cytosol from mitochondria and in turn promotes caspase activation by proteolyzing inhibitor of apoptosis proteins. Here we asked whether treatment with an HtrA2/Omi inhibitor, 5-[5-(2-nitrophenyl)furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid (UCF-101), restores heart dysfunction following ischemia/reperfusion injury in vivo. Rats underwent a 30-min ischemia by occluding the left anterior descending artery, followed by 24 h reperfusion. UCF-101 (0.75 or 1.5 μmol/kg, i.p.) was administered 10 min before reperfusion. UCF-101 treatment significantly recovered the mean arterial blood pressure and ameliorated contractile dysfunction of the left ventricle 72 h after reperfusion with concomitant reduction of infarct size. Cardio-protection mediated by UCF-101 was correlated with reduced X-linked inhibitor of apoptosis protein (XIAP) degradation and inhibition of Caspase-9, Caspase-3, and Caspase-7 processing. Furthermore, UCF-101 prevented loss of membrane integrity by inhibiting fodrin breakdown in cardiomyocytes. UCF-101-induced cytoprotection was also correlated with reduced Fas ligand expression and inhibition of FLIP degradation following ischemia/reperfusion. These results suggest that UCF-101 rescues cardiomyocytes from ischemia/reperfusion injury by inhibiting XIAP degradation and Fas/Fas-ligand-induced apoptosis, thereby ameliorating ischemia/reperfusion-induced myocardial dysfunction.

Introduction

Growing evidence from animal experiments and clinical observations indicates that myocardial infarction after ischemia/reperfusion is a consequence not only of necrosis but also apoptosis (Eefting et al., 2004). Recent observations by different researchers demonstrate that blocking signal transduction leading to apoptosis significantly reduces myocardial infarct size and ameliorates cardiac function (Gao et al., 2002, Misra et al., 2003, Lee et al., 2003). Notably, the apoptosis pathway contributes in part to myocardial ischemia/reperfusion-induced injury, and therefore anti-apoptotic treatments may improve clinical outcomes in patients with ischemic heart disease.

The central players in apoptotic pathways are caspases, members of the cysteinyl aspartate family of proteases. Caspases are activated in response to diverse stimuli and cleave multiple cellular substrates, resulting in cell death (Earnshaw et al., 1999). The main endogenous inhibitors of caspases are members of the inhibitor of apoptosis family proteins, such as X-chromosome linked inhibitor of apoptosis protein (XIAP) (Deveraux and Reed, 1999). Recent studies demonstrate that during induction of apoptosis, the mitochondrial protein high temperature requirement A2 (HtrA2)/Omi is released into the cytoplasm concurrent with cytochrome c and cleaves XIAP, resulting in Caspase-9, Caspase-3 and Caspase-7 activation (Deveraux and Reed, 1999, Suzuki et al., 2001, Hegde et al., 2002, Martins et al., 2002, Verhagen et al., 2002). The HtrA2/Omi precursor is a 50-kDa protein whose N-terminal peptides are cleaved after mitochondrial transport, thereby generating a 36-kDa mature protein with an N-terminus resembling the second mitochondria-derived activator of caspase (SMAC). The mature form of HtrA2/Omi but not its precursor binds to inhibitor of apoptosis proteins in a manner similar to SMAC. HtrA2/Omi also induces cell death in mammalian cells independent of caspases, Apaf-1 or inhibitor of apoptosis proteins via its serine protease activity (Hegde et al., 2002). HtrA2/Omi exhibits a PDZ domain, a protein–protein interaction module recognizing specific C-terminal sequences in membrane receptors and ion channels (Harris and Lim, 2001). The HtrA2/Omi PDZ domain serves as a sensitive regulator of its protease activity.

The Fas (CD95)/Fas ligand (CD95L) system is a key regulator of apoptosis. Fas is a 48-kDa transmembrane receptor glycoprotein, and its expression is enhanced in cardiomyocytes exposed to hypoxia (Tanaka et al., 1994, Date et al., 2003). The Fas ligand (FasL), which binds to the Fas receptor, belongs to the tumor necrosis factor superfamily of cytokines and can exist as a membrane-bound or a soluble protein. Binding of FasL to Fas leads recruits the Fas-associated death domain adapter molecule to the receptor complex, which includes procaspase-8, thereby promoting procaspase-8 activation by autoproteolysis (Varfolomeev et al., 1998). Subsequently downstream caspases including Caspase-3 are activated followed by cleavage of key regulatory proteins resulting in apoptosis (Varfolomeev et al., 1998). Recently, the FLICE-inhibitory protein (FLIP), which is homologous to Caspase-8 (FLICE), was identified as an anti-apoptotic protein (Thome et al., 1997). FLIP is highly expressed in the heart under normal physiological conditions but is degraded after ischemia/reperfusion (Rasper et al., 1998). Activated monocytes, which release soluble FasL after stimulation, induce death of neonatal rat cardiomyocytes via the Fas-mediated pathway (Imanishi et al., 2000). These results suggest that the Fas-mediated cell death pathway functions in cardiomyocytes but is down-regulated by inhibitors such as FLIP under normal conditions. After stress such as ischemia, however, FLIP becomes inactivated, leaving cells susceptible to death via the Fas-mediated pathway.

Recently Cilenti et al. (2003) reported a specific inhibitor of HtrA2/Omi, 5-[5-(2-nitrophenyl)furfuryliodine]1,3-diphenyl-2-thiobarbituric acid (UCF-101), which significantly inhibits apoptotic cell death in Caspase-9 null fibroblasts. Liu et al. (2005) also documented that UCF-101 rescue cardiomyocytes from ischemia/reperfusion injury by inhibiting XIAP degradation. However, a potential effect on heart dysfunction in heart ischemia/reperfusion following UCF-101 treatment was not addressed. It is also unclear whether UCF-101 inhibits a Caspase-9-independent apoptotic pathway such as Fas/FasL. We here used a rat myocardial ischemia/reperfusion to investigate whether UCF-101 treatment ameliorates ischemia-induced heart dysfunction. We also asked whether UCF-101 inhibits Fas/FasL-dependent apoptotic pathway in ischemic cardiomyocytes.

Section snippets

Materials

A polyclonal antibody against the 150-kDa fodrin breakdown product was a gift of Dr. Y. Shirasaki (Daiichi Pharmaceutical Co., Ltd., Tokyo, Japan) (Sato et al., 1999). The following reagents and antibodies were obtained from the indicated sources: 5-[5-(2-nitrophenyl)furfuryliodine]-1,3-diphenyl-2-thiobarbituric acid (UCF-101) (Calbiochem, Darmstadt, Germany); 2,3,5-triphenyltetrazolium chloride (TTC) and Evans blue (Sigma-Aldrich, St Louis, MO USA); anti-HtrA2/Omi antibody (R&D Systems,

HtrA2/Omi inhibition ameliorates myocardial injury after myocardial ischemia/reperfusion

To determine whether inhibiting HtrA2/Omi has cardioprotective effect, a group of rats was subjected to 30 min ischemia and 24 h of reperfusion, and the effect of UCF-101, a novel, specific inhibitor of HtrA2/Omi protease activity, on myocardial infarct size was determined. There was no significant difference in the volume of risk areas between vehicle- and UCF-101-treated animals (Fig. 1A and B), indicating that a comparable degree of ischemia was induced in both groups. However, UCF-101

Discussion

This study was designed to evaluate potential protective effects of the HtrA2/Omi inhibitor, UCF-101, on ischemia/reperfusion-induced cardiac dysfunction after a brief period of ischemia/reperfusion. We confirmed a previous study (Liu et al., 2005) in which UCF-101 treatment before reperfusion provided marked protection of heart tissue against ischemia/reperfusion-induced infarction. We show here that recovery of cardiac contractile function is positively correlated with the prevention of

Acknowledgement

This work was supported in part by grants-in-aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan (14370035 to K.F.).

References (38)

  • T. Sato et al.

    DY-9760e, a novel calmodulin antagonist, reduces brain damage induced by transient focal cerebral ischemia

    Eur. J. Pharmacol.

    (1999)
  • Y. Suzuki et al.

    A serine protease, HtrA2, is released from the mitochondria and interacts with XIAP, inducing cell death

    Mol. Cell

    (2001)
  • A. Trencia et al.

    Omi/HtrA2 promotes cell death by binding and degrading the anti-apoptotic protein ped/pea-15

    J. Biol. Chem.

    (2004)
  • E.E. Varfolomeev et al.

    Targeted disruption of the mouse Caspase 8 gene ablates cell death induction by the TNF receptors, Fas/Apo1, and DR3 and is lethal prenatally

    Immunity

    (1998)
  • A.M. Verhagen et al.

    HtrA2 promotes cell death through its serine protease activity and its ability to antagonize inhibitor of apoptosis proteins

    J. Biol. Chem.

    (2002)
  • K. Wang et al.

    Simultaneous degradation of alpha II- and beta II-spectrin by caspase-3 (CPP32) in apoptotic cells

    J. Biol. Chem.

    (1998)
  • Q.L. Deveraux et al.

    IAP family protein's suppressors of apoptosis

    Genes Dev.

    (1999)
  • W.C. Earnshaw et al.

    Mammalian Caspases: structure, activation, substrates, and functions during apoptosis

    Ann. Rev. Biochem.

    (1999)
  • F. Eefting et al.

    Role of apoptosis in reperfusion injury

    Cardiovasc. Res.

    (2004)
  • Cited by (0)

    View full text