Antioxidative action of N--tosyl-L-lysine chloromethyl ketone prevents death of glutathione-depleted cardiomyocytes induced by hydrogen peroxide

Hydrogen peroxide (H2O2) induces the hypertrophy in cultured H9c2 cardiomyocytes and cell death in glutathione (GSH)-depleted H9c2 cells. In the present study, we observed that pretreatment with a serine protease inhibitor, N--tosyl-L-lysine chloromethyl ketone (TLCK), significantly prevented the H2O2-induced cell damages in GSH-depleted H9c2 cells in a concentration-dependent manner. The phase contrast microscopy revealed that although the exposure of the GSH-depleted H9c2 cells to H2O2 resulted in a globular shape of the cells, TLCK prevented the occurrence of H2O2-induced morphological changes. TLCK also inhibited the generation of reactive oxygen species in the cells after addition of H2O2, suggesting that the antioxidant action of TLCK is involved in the protection against the cell damages by H2O2. Application of TLCK after ~30 min of exposure to H2O2 could significantly protect the cells from cell damages. The other serine protease inhibitors that were tested could not prevent the cell damages in GSHdepleted H9c2 cells. Pretreatment with an inhibitor of nuclear factor- translocation into the nucleus and a proteasome inhibitor did not prevent the cell damages in GSH-depleted H9c2 cells. An inhibitor of p53 significantly prevented the cell damages in GSH-depleted H9c2 cells. These results suggest that antioxidative action of TLCK prevents the death of GSH-depleted H9c2 cardiomyocytes induced by H2O2.