Hydrogen peroxide predisposes neonatal rat ventricular myocytes to Fas-mediated apoptosis.
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 h to recombinant Fas ligand (rFasL, 10 ng/ml) plus an enhancing antibody (1 microg/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 FLIP(L) 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.
Yaniv
Gal
G
Rappaport Family Institute for Research in the Medical Sciences, The Ruth and Bruce Rappaport Faculty of Medicine, Technion-Israel Institute of Technology, Haifa, Israel.
Shilkrut
Mark
M
Larisch
Sarit
S
Binah
Ofer
O
D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis; D002478:Cells, Cultured; D004357:Drug Synergism; D053222:Fas Ligand Protein; D006352:Heart Ventricles / Q000166:cytology; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000494:pharmacology*; D032383:Myocytes, Cardiac / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction / Q000187:drug effects
D053222:Fas Ligand Protein; C505662:Faslg protein, rat; D008562:Membrane Glycoproteins; D006861:Hydrogen Peroxide