Abstract 234: Myocardial Ischemia-Reperfusion Injury Causes Selective Rather Than Global Inhibition of Proteasomal Function
Decreased proteasomal function has recently been identified as one of the contributors towards ischemia-reperfusion injury of the myocardium. However, the extent of proteasomal inactivity and its role in ischemic injury has yet to be ascertained. As proteasomal degradation plays a key role in modulating gene regulatory activities, we have monitored the degradation of phospho-IkB (NFkB), Keap-1 (Nrf-2) and phospho-FOXO1 for assessing proteasomal function in ischemic-reperfused myocardium. Ischemic injury was induced in Group-I rats by left anterior descending coronary artery (LAD) occlusion for 30, 60 and 120 min while for the Group-IR, 30 min of ischemia was followed by 60, 120 and 240 min reperfusion. Group-PC rats were subjected to four repetitive cycles of 5 min ischemia followed by 10 min of reperfusion, then 30 min ischemia and 60, 120 and 240 min reperfusion. Ischemic zone of the hearts was then used for analysis. Proteasomal inactivity was first assessed by monitoring the transcript level of GRP78, a hallmark of Unfolded Protein Response, one of the major consequences of proteasomal inactivation. Northern analysis revealed that during ischemia-reperfusion, GRP78 mRNA level rapidly diminishes followed by a robust induction. Western immuno-blotting results showed that with progressive ischemia, degradation of phosphorylated-IkB (p-IkB) decreased, resulting in an increase in p-IkB to IkB ratio. In reperfused myocardium, such accumulation of p-IkB was further accentuated while in preconditioned-reperfused heart, p-IkB to IkB ratio was comparable to that in normal. Involvement of proteasome in maintaining p-IkB level was confirmed by its accumulation in H9c2 myoblasts treated with LPS and proteasomal inhibitor lactacystin. Furthermore, on the contrary to p-IkB accumulation, degradation of Keap-1 remained unhindered both during ischemia and reperfusion. Also, neither ischemia nor reperfusion caused any significant modulation of FOXO-1, except inducing its rapid translocation from the nucleus to the cytoplasm. Taken together, it is concluded that ischemia-reperfusion injury induces selective rather than global inhibition of proteasomal function and thus it affects only a set of gene regulatory events while sustaining the others.