Neonatal rat ventricular myocytes (NRVM) grown in normoxic environment are not susceptible to Fas-induced apoptosis. In the present work, we tested the hypothesis that free radical injury represented by transient exposure to H2O2 sensitizes NRVM to Fas-mediated apoptosis. NRVM were treated with H2O2 (0.5 mM) for 2-4 h and thereafter exposed for 7 It to recombinant Fas ligand (rFasL, 10 ng/ml) plus an enhancing antibody (1 mu g/ml). Apoptotic cardiomyocytes were counted and apoptosis-related proteins were measured by Western blot. H2O2 alone induced apoptosis (9.4 +/- 1.0%) that was preceded by activation of caspases-8 and -3, and PARP degradation. Incubation of NRVM with H2O2, followed by exposure to rFasL, increased the apoptotic index to 13.8 +/- 2.0%, but did not change caspase-8 or PARP activation. To investigate the mechanism underlying the sensitizing affect of H2O2 towards Fas-induced apoptosis, we studied the effects of H2O2 on the expression of key apoptosis signaling proteins. Incubation with H2O2 for 2-4 h decreased Fas expression and the expression of the Fas-related antiapoptotic proteins FLIPL and ARC, and increased the expression of the antiapoptotic proteins bcl-2 and xIAP. FADD expression was unchanged. Next, we tested the effect of H2O2 on the apoptosis-inducing, Fas-dependent Daxx-ASK-1-JUN kinase pathway. H2O2 dramatically increased ASK-1 expression and JUN kinase activation, but did not effect Daxx expression. Based on these findings we concluded that H2O2 sensitizes NRVM to Fas-mediated apoptosis by activating the Daxx-ASK-1-JUN kinase pathway, and by shifting the balance between proapoptotic and antiapoptotic proteins towards the former. (c) 2005 Elsevier Inc. All rights reserved.