Abstract P271: Genetically Induced Moderate Inhibition of the Proteasome in Cardiomyocytes Exacerbates Myocardial Ischemia-Reperfusion Injury in Mice

Abstract

Proteasome dysfunction is implicated human ischemic heart disease and observed in experimental myocardial ischemia-reperfusion (I/R) injury. Altered proteasome activities in I/R hearts were reported but it is unclear whether proteasome function in I/R hearts is adequate. Moreover, effects of pharmacological inhibition of the proteasome on I/R injury remain controversial. Hence we sought to determine the adequacy of proteasomal function in I/R hearts and the impact of moderate cardiomyocyte-restricted proteasome inhibition (CR-PSMI) on I/R injury in intact animals. First, myocardial I/R was created by ligation (30min) and subsequent release of the left anterior descending artery in the transgenic (tg) mice overexpressing GFPdgn, a previously validated surrogate proteasome substrate. Compared with the sham controls, myocardial GFPdgn protein levels in the remote area, the board zone, and the a rea a t r isk (AAR) of the ventricle 24hrs after reperfusion were significantly increased, indicative of proteasome functional insufficiency (PFI) in the I/R heart. The most important proteasome peptidase resides in β5 subunits of the 20S proteasome. The clinically used proteasome inhibitor bortezomib targets specially the β5 subunit. To achieve CR-PSMI in intact animals, we engineered and tested a catalytically inactive mouse β5 subunit mutant (T60A-β5) and created multiple tg mouse lines in which T60A-β5 was overexpressed under the control of an attenuated mouse mhc6 promoter. Baseline analyses of the tg lines confirm the ability of T60A-β5 to replace endogenous β5 and inhibit proteasome chymotrypsin-like activities in the heart in a dose-dependent manner. A stable tg line with ∼40% replacement was used in this study. The same I/R procedure caused a significantly greater infarct size in T60A-β5 tg mice (66.2% of AAR) than in the littermate Ntg mice (53.4% of AAR). Under either the sham surgery or the I/R condition, a decrease in phosphorylated AKT and an increase in PKCδ proteins were evident in the tg hearts, compared with the respective Ntg groups. These results show that I/R causes PFI in the heart and PFI plays a pathogenic role in I/R injury. A potential mechanism by which PFI contributes to I/R injury is to suppress AKT activation and exacerbate PKCδ signaling.