ROS-mecliated ERK activation in delayed protection from anoxic preconditioning in neonatal rat cardiomyocytes

摘要

Background The activation of extracellular signal-regulated kinase1/2 (ERK_(1/2)) has been shown to be important signaling pathway in the ischemic preconditioning (IPC) response. Recently, some studies suggest a key role for the mitochondrial ATP-sensitive potassium channel (mK_(ATP)) as both a trigger and an end effector of acute and delayed protection of IPC. Hence, this study was undertaken to elucidate the relationship between mK_(ATP) and ERK_(1/2) in the delayed protection mechanism of anoxic preconditioning (APC). Methods An APC model was established using cultured neonatal rat cardiomyocytes. Pharmacological agents [ diazoxide, 5-hydroxydecanoate ( 5-HD ), 2-mercaptopropionylglycine (MPG), and PD98059] were used to modulate mK_(ATP) and ERK_(1/2) activation. Cellular injury was evaluated by measuring cellular superoxide dismutase (SOD) activity, cell viability, and lactate dehydrogenase (LDH) release. The generation of cellular reactive oxygen species (ROS) and the activation of ERK_(1/2) were determined at different time points starting from the beginning of preconditioning with anoxia or diazoxide (an mK_(ATP) opener). Results Cell viability and SOD activity in the APC [(81. 9 +-11. 4)%, (13.6 +- 3.7) U/L] and diazoxide [(79. 2 +-12. 4)% , (16. 5 +-4. 6) U/L] groups were significantly higher than in the anoxia/reoxygenation (A/R) [(42.2 +-7.3)%, (8.8+-2.8) U/L] group (all P<0.01). LDH activity in the APC group [ (101.9 +-18.9) U/L] and diazoxide group [(97.5 +- 17.7) U/L] was significantly tower than in the A/R group [(250.5 +- 43.6) U/L] (all P<0.01). Both APC and diazoxide simultaneously facilitated intracellular ROS generation and rapid ERK_(1/2) activation. But the effects of APC and diazoxide were remarkedly attenuated by 5-HP (an mK_(ATP) blocker) and by MPG (a free radical scavenger). In addition, the ERK_(1/2) inhibitor PD98059 also abolished the cellular protective effects induced by diazoxide. Conclusion mK_(ATP) may mediate ERK_(1/2) activation during anoxia preconditioning by generating ROS, which then triggers the delayed protection of APC in rat cardiomyocytes.