Abstract 266: Truncated Glycogen Synthase Kinase 3β Increases Mitochondrial Fragmentation, Reactive Oxygen Species generation, and Cell Injury

Introduction: Oxidative stress during ischemia reperfusion (IR) injury remains a major obstacle during percutaneous coronary intervention after acute myocardial infarction. It often leads to an increased susceptibility for mitochondria permeability transition pore (mPTP) opening leading to cell death and worsened contractile recovery. Glycogen synthase kinase 3β (GSK-3β) is proposed as one of the key molecules that regulate mitochondrial dysfunction and injury during IR. Oxidative stress increases activation of matrix metalloproteinase 2 (MMP2) and subsequent cleavage (amino acids 1-34) of GSK-3ß to form a constitutively-active truncated form. However, the molecular mechanism underlying how GSK-3β activity controls mitochondrial form/function under oxidative stress condition is not fully understood. Hypothesis: Oxidative stress induces GSK-3ß to form a constitutively-active truncated form through MMP2-mediated cleavage, which leads to increased susceptibility of mPTP opening and activation of cell death signaling. Methods: Using overexpression of full length and truncated GSK-3β fused to GFP in H9C2 cells we determined the effects of GSK-3β on mitochondrial morphology and reactive oxygen species (ROS) generation using confocal microscopy. Activation of cell death signaling was evaluated by cytochrome c release, caspase activation, and GSK-3β activity using Western blotting. Results: Non-transfected and full-length GSK-3ß had comparable mitochondrial networking and morphology whereas cells overexpressing truncated GSK-3ß showed distinct mitochondrial fragmentation and swelling. Similarly, the non-transfected and full-length GSK-3ß overexpressing cells had equivalent levels of ROS whereas the truncated-GSK-3ß overexpressing cells had markedly increased levels of basal ROS that was further enhanced with transient H2O2 treatment. Cells overexpressing truncated GSK-3ß had an increased susceptibility to mPTP opening as measured by cytochrome C release to the cytoplasm when challenged with H2O2. Conclusion: Truncation of GSK-3ß induces increased mitochondrial ROS formation as well as mitochondrial fragmentation that is indicative of mitochondrial damage and an increased susceptibility to mPTP opening.