Abstract 217: Hexokinase II Binding to Mitochondria Suppresses Irreversible Ischemia Reperfusion Injury in the Beating Heart by Respiratory Inhibition and Reduced ROS Levels

Abstract

Introduction: The glycolytic enzyme hexokinase II (HKII) can be mitochondrial-bound (mtHK) or free in the cytosol. Increased mtHKII protects the heart against ischemia-reperfusion (I/R) injury. The mechanism of protection is not yet elucidated. Hypothesis: We hypothesized that mtHKII protects the heart against I/R injury by respiratory inhibition and reducing reactive oxygen species (ROS). Methods: Langendorff perfused rat hearts were exposed for 20min to 1 μ M TAT-only, 1 μ M TAT-HK or 200 nM TAT-HK, followed by 15 min ischemia and 30 min reperfusion. TAT-HK contains the binding motif of HKII and dislodges HKII from mitochondria. ROS was measured using DHE fluorescence (n=6/group). Lactate was measured after peptide treatment, necrosis (LDH release) was determined during reperfusion and oxygen consumption (MVO 2 ) and heart function (rate pressure product (RPP)) were monitored (n=12-15/group). Results: Disruption of mtHKII binding resulted in increased MVO 2 /RPP values during both baseline and reperfusion in the 1 μ M TAT-HK group. Effluent lactate increased from (mean±SEM) 0.024±0.0041 μ mol/ml effluent in the TAT-only group to 0.042±0.0072 and 0.091±0.016 μ mol/ml effluent in the 200 nM and 1 μ M TAT-HK treated hearts, respectively. TAT-HK treatment had no effect on ROS production during baseline conditions. However, during both ischemia (normalized values (mean±SEM) 1.14±0.05 and 1.17±0.08 versus 1.09±0.06) and reperfusion (1.58±0.24 and 1.84±0.15 versus 1.30±0.17) TAT-HK significantly and dose dependently increased ROS when compared to TAT-only treatment. This was accompanied by an increased LDH release during reperfusion of (mean±SEM) 27.1±4.1 and 42.9±4.2 μ mol/30min/g heart for the 200 nM and 1 μ M TAT-HK treated hearts, respectively. There was no cell necrosis in the TAT-only group. Conclusion: >Our results show for the first time that disruption of the mtHK binding affects cardiac MVO 2 and causes an increase in ROS production during ischemia and reperfusion in the intact heart. These data indicate that mtHKII is a master switch turning reversible ischemia into irreversible ischemia. Our data also suggest that mtHKII is a determinant of glycolysis/glucose oxidation coupling. Supported by the Dutch Heart Foundation (NHS 2010B011)