translocation of Ref-1, which is almost completely inhibitedwhen the hearts were pretreated with antisense Ref-1 (as-Ref-1)that also abolished the cardioprotective adaptive response. Sig-nificant amount of NFκB and Nrf2 was found to be associatedwith Ref-1 in the nuclear extract obtained from the left ventricleimmunoprecipitated with Ref 1. Such Ref-1-NFκB and Ref-1-Nrf2 interactions were significantly inhibited with as-Ref-1.However, the association of Trx-1 with NFκB is increased in theadapted heart, which was significantly blocked by as-Ref-1.Nrf2 was also associated with NFκB; however such associationappeared to be independent of Ref-1. In contrast, myocardialadaptation to ischemia inhibited the ischemia reperfusion-induced loss of Nrf2 from the nucleus, which was inhibited byas-Ref-1. Cardiac adaptation induced survival signal (phos-phorylation of Akt) was also inhibited by as-Ref-1. Confocalmicroscopy confirmed the results of immunoblotting clearlyshowing the nuclear translocation of Ref-1 and nuclear 3D co-localization of Ref-1 with NFκB in the adapted heart and itsinhibition with as-Ref-1. Our results show that preconditioningpotentiates a survival signal by causing nuclear translocationand activation of Ref-1, where significant interaction betweenNFκB and Ref-1, Trx-1 and Nrf2 appears to regulate Ref-1-induced survival signal.

Keywords: Ref-1; Cardiac adaptation; Ischemia/reperfusioninjury

doi:10.1016/j.yjmcc.2007.03.573

Signalling mechanisms and phospholipase A2 translocationin TNF-α mediated cytoprotection in ischaemiaBenjamin Loos, Robert M. Smith, Anna-Mart Engelbrecht.Dept. of Physiological Sciences, Stellenbosch University,South Africa

Tumor Necrosis Factor-α (TNF-α) and the cytosolic phos-pholipase A2 (cPLA2) are involved in the cellular inflammatoryresponse and ischaemic injury. During myocardial ischaemiaTNF-α is endogenously generated, activating cPLA2. Inaddition, low dose TNF-α confers cytoprotection. cPLA2 acti-vation results in the generation of mediators of cellular injury,apoptosis and necrosis. We have therefore investigated the roleof cPLA2 in TNF-α mediated cytoprotection. Murine C2C12myoblasts were grown to 70% confluency and differentiatedinto myotubes. Myotubes were then included in one of thefollowing experimental groups: normoxic control; ischaemiccontrol; ischaemic preconditioning (IPC); TNF-α precondition-ing (TNF-PC); and TNF-PC with AACOCF3 (cPLA2 inhibitor,TNF-I). Following an ischaemic insult, cell viability was deter-mined. In addition cPLA2 phosphorylation state and cellularlocalisation was assessed using western blotting and fluores-cence immunohistochemistry combined with 3-D imagingtechniques and analysis. IPC, TNF-PC and TNF-I preventedthe ischaemia induced decrease in cell viability. cPLA2 phos-phorylation was absent in the IPC group, and present at about

40% of control levels in the TNF-PC and TNF-I groups fol-lowing the ischaemic insult. Cellular localisation of cPLA2

showed distinct differences between the IPC, TNF-PC andischaemic control groups. This data suggests an additional levelof regulation in the TNF–cPLA2 axis, with a possible role forcPLA2 in deciding cell fate.

Keywords: Ischaemic injury; Tumour necrosis factor alpha(TNF-α); Cytosolic Phospholipase A2 (cPLA2)

doi:10.1016/j.yjmcc.2007.03.574

Sulforaphane in the protection of cardiomyocytes fromoxidative stressMarco Malaguti, Cristina Angeloni, Emanuela Leoncini,Eleonora Pagnotta, Pierluigi Biagi, Silvana Hrelia. Dept. ofBioch., Univ. Bologna, Italy

The recognition that oxidative stress plays a major role in thepathophysiology of cardiac disorders has led to extensiveinvestigation of the protective effects of exogenous antioxidants.Another strategy may be through induction of the endogenousantioxidants/phase 2 enzymes. Accordingly, this study wasundertaken to investigate the inducibility of endogenous antio-xidants/phase 2 enzymes (reduced glutathione (GSH), GSHperoxidase (GPx), glutathione reductase (GR), GSH S-transfer-ase (GST), and NAD(P)H:quinone oxidoreductase 1 (NQO1)) bythe isothiocyanate sulforaphane (SF) in cultured rat cardio-myocytes. Due to the role of the nuclear factors E2-related factor1 (nrf1) and 2 (nrf2) in inducing phase 2 enzymes, we alsoinvestigated the role of SF in the translocation of these factorsfrom cytosol to the nucleus. Enzymatic activities were deter-mined spectrophotometrically, GSH by the fluorescent indicatormonochlorobimane, reactive oxygen species (ROS) formationspectrofluorimetrically by 2′,7′-dichlorofluorescein diacetate, cellviability by MTT assay, nrf1 and nrf2 translocation to nucleus bylaser confocal microscopy. GPx activity was not affected by SF,but GR, GSTand NQO1 activities and GSH levels increased witha time-dependent pattern following SF supplementation. As aconsequence SF supplementation led to decreased ROS levelsand increased cell viability following oxidative stress. Laserconfocal microscopy revealed the translocation of both nrf1 andnrf2 to the nucleus. In conclusion, SF acts as an indirectantioxidant leading to cardioprotection against oxidative stress.

Keywords: Antioxidants; Cardiomyocytes; Enzymes

doi:10.1016/j.yjmcc.2007.03.575

Subcellular redistribution of protein kinase c in chronicallyhypoxic rat heartMarketa Hlavackova1, Jan Neckar2,3, Olga Novakova1,3,Frantisek Kolar2,3, Rene Musters4, Frantisek Novak1. 1FacSci, Charles Univ, Prague, Czech Republic. 2Inst Physiol, Acad

S188 ABSTRACTS / Journal of Molecular and Cellular Cardiology 42 (2007) S171–S189