Abstract 12375: Inhibition of ERS Leads to Cardioprotection against Ischemia Reperfusion Injury by Preventing the mPTP Opening
While it is well known that endoplasmic reticulum stress (ERS) plays an important role in myocardial ischemia/reperfusion (I/R) injury and inhibition of ERS leads to cardioprotection against I/R injury, the precise mechanism by which inhibition of ERS induces cardioprotection remains unclear. We aimed to test if inhibition of ERS can prevent the mitochondrial permeability transition pore (mPTP) opening and to explore the signaling mechanism whereby ERS inhibition leads to the protection against the mPTP opening. Exposure of cardiac H9c2 cells to 800 µM H2O2 for 20 min caused a marked decrease in TMRE fluorescence, indicating mPTP opening by oxidative stress. Compared to the control, 20, 30, and 40 µM TUDCA prevented the loss of TMRE fluorescence (63.2±4.8%, 75.3±2.7%, 63.6±2.4%), indicating that inhibition of ERS leads to the prevention of mPTP opening. The effect of TUDCA on TMRE fluorescence was inhibited by the PI3K inhibitor LY294002 and the PKG inhibitor KT5823, implying that the PI3K/Akt and PKG signaling pathways may mediate the action of TUDCA. TUDCA at different concentrations significantly increased GSK-3β phosphorylation at Ser9 with the pick at 30 µM (285.6 ± 9.9%). The expression level of GRP78 was also most prominent with 30 µM TUDCA (37.3 ± 5.7%). TUDCA-induced increases in Akt and GSK-3β phosphorylation were inhibited by LY294002, whereas KT5823 could suppress phosphorylation of VASP and GSK-3β by TUDCA. Experiments with transmission electron microscopy revealed that TUDCA prevented H2O2-induced swelling of endoplasmic reticulum and mitochondrial damages. Studies with flow cytometry showed that TUDCA given at reperfusion increased cell viability (78.8 ± 3.9 % vs. 55.3 ± 2.9 % in control) in cells subjected to simulated ischemia/reperfusion, indicating that TUDCA can prevent reperfusion injury. However, TUDCA was not able to increase cell viability in cells transfected with the constitutively active GSK-3β (GSK-3β-S9A-HA) mutant, strongly supporting the essential role of GSK-3β inactivation in cardioprotection. In conclusion, these data suggest that inhibition of ERS leads to the prevention of mPTP opening and reperfusion injury through inactivation of GSK-3β. The PI3K/Akt and PKG pathways may mediate inactivation of GSK-3β.
- © 2012 by American Heart Association, Inc.