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 H 2O2 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 μg/ml). Apoptotic cardiomyocytes were counted and apoptosis-related proteins were measured by Western blot. H2O 2 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.
|Number of pages||7|
|Journal||Biochemical and Biophysical Research Communications|
|State||Published - 28 Oct 2005|
Bibliographical noteFunding Information:
The technical assistance of Ms. Irina Reiter is greatly appreciated. This work was supported by Servier through the Center of Molecular Therapeutics at Columbia University—New York, the Israel Academy of Sciences, Minerva Foundation through the Bernard Katz Center for Cell Biophysics, and the Rappaport Family Institute for Research in the Medical sciences.
- Hydrogen peroxide
ASJC Scopus subject areas
- Molecular Biology
- Cell Biology