Role of Nrf1 and Nrf2 in H2O2 preconditioning in cardiac cells.

Ischemic preconditioning is a cardioprotective phenomenon that involves adaptive changes in cells and molecules. This adaptation occurs in a biphasic pattern: an early phase (1-2h), and a late phase (12-24h). While it is widely accepted that reactive oxygen species (ROS) are strongly involved in triggering ischemic preconditiong, it is not clear if they play a major role in the early or late phase of preconditioning and which are the mechanisms involved. The present study was designed to investigate the mechanisms behind H2O2 induced cardioprotection in cardiac cells focusing on the induction of phase II enzymes and the involvement of Nuclear factor E2-related factor 1 (Nrf1) and Nrf2. Rat neonatal cardiomyocytes were grown until confluence. Preconditioning was simulated with 100 µM H2O2 for 10 min and after different times oxidative stress was generated by 100 µM H2O2 for 30 min. Cell viability was evaluated by MTT, and ROS production by 2′,7′-dichlorofluorescein-diacetate. Phase II enzyme activities were determined spectrophotometrically, and Nrf1 and Nrf2 nuclear translocation by immunoblotting. To evaluate the involvement in phase II enzyme induction, Nrf1 and Nrf2 were silenced with Nrf1-siRNA and Nrf2-siRNA. H2O2-preconditioning was more effective against oxidative stress in the late than in the early phase of adaptation. Surprisingly, H2O2-preconditioning led to a decrease in intracellular ROS production consistent with the induction of glutathione reductase, catalase, thioredoxine reductase and NADPH quinone oxidoreductuase 1. H2O2-preconditioning induced the nuclear translocation of both Nrf1 and Nrf2. The down-regulation of Nrf1 and Nrf2 by Nrf1-siRNA and Nrf2-siRNA partially inhibited the enzyme induction. These findings demonstrate a key role for Nrf1, and not only for Nrf2, in the induction of phase II enzymes following H2O2-preconditioning