Abstract 67: Inhibition of Myocardial Apoptosis by Postconditioning Is Associated with Attenuation of Oxidative Stress-Mediated NFκB Translocation and Cytokine Release
Background: Oxidative stress stimulated-NFκB activation has been associated with rapid transcription of cytokines and induction of apoptosis. This study tested the hypothesis that postconditioning (Postcon) attenuates myocardial apoptosis by inhibiting translocation of NFκB and release of cytokines secondary to a reduction of oxidative stress during reperfusion (R).
Methods and results: Rats were subjected to 30 min ischemia and 3 h of R. Relative to Control (n=10), Postcon (n=10) with three cycles of 10 s R and 10 s re-occlusion applied at the onset of R reduced the plasma malondialdehyde (0.8±0.06* vs. 1.21±0.08 μM/ml), consistent with a decrease in superoxide generation (DHE staining) in area at risk myocardium at 30 min of R. Expression of NFκB was reduced (99±4* vs. 148±13%) and its translocation to nuclei was inhibited (142±18* vs. 167±21%) with Postcon compared to control. At 60 min of R, elevated plasma TNFα (667±130* vs. 1994±447 pg/ml) and IL6 (1725±455* vs. 2688±643 pg/ml) observed in control were attenuated with Postcon. These results were consistent with reduced positive TNFα and IL6 staining detected by immunohistochemistry in myocardium, At the end of 3 h of R, the number of apoptotic cells (% total nuclei) in ischemic myocardium was reduced by Postcon (11±2* vs. 20±1), with attenuated appearance of DNA ladders versus control. Caspase-3 activity was inhibited (0.21±0.1* vs. 0.57±0.1%) and Bcl-2 expression was increased (138±10* vs. 100% of Control) in Postcon. To directly demonstrate if oxidants induce apoptosis by NFκB-cytokine signaling pathway, N-acetylcysteine (NAC, n=10), a potent anti-oxidant agent, was given intravenously. NAC inhibited NFκB expression (42±8*) and reduced release of TNFα (231±72*) and IL6 (666±300*) to a comparable level with Postcon. In addition, apoptotic cells (12±1%*) and the caspase-3 activity (0.33±0.1%*) were comparably reduced, and Bcl-2 expression was increased (135±10%*) by NAC.
Conclusion: These data suggest that Postcon attenuates oxidant-induced apoptosis, inhibits caspase-3 activity and augments expression of pro-apoptotic protein, Bcl-2, potentially mediated by altering NFκB-cytokine signaling pathways. *p<0.05 Postcon and NAC vs. Control.