Abstract 1602: Inhibition of GSK Can Produce Cardioprotection by Slowing Adenine Nucleotide Transport across the Outer Mitochondrial Membrane without Affecting the Mitochondrial Permeability Transition
Inhibition of GSK-3b has been shown to reduce ischemia-reperfusion injury by mechanisms that involve the mitochondria. The goal of this study was to determine the molecular targets and mechanistic basis of this cardioprotective effect. In perfused rat hearts, treatment with GSK-3b inhibitors (GSKi) prior to 20 min of ischemia, significantly improved recovery of function. To assess the effect of GSKi on mitochondrial function under ischemic conditions, mitochondria were isolated from rat hearts perfused with GSKi and treated with uncoupler/ cyanide or were made anoxic by allowing the mitochondria to consume all of the oxygen in a sealed chamber. We found that GSKi slowed ATP consumption under these conditions. This reduction in ATP consumption could be due to inhibition of ATP entry into the mitochondria through VDAC and/or ANT or to inhibition of the F1F0ATPase. To determine the site of the inhibitory effect on ATP consumption, we measured the conversion of ADP to AMP by adenylate kinase located in the intermembrane space. This assay requires adenine nucleotide transport across the outer mitochondrial membrane but not transport across the inner membrane or metabolism in the matrix. We found that GSKi slow AMP production. This suggests that GSKi are acting on outer mitochondrial membrane transport. In sonicated mitochondria, GSKi had no effect on ATP consumption. In intact mitochondria, cyclosporin A had no effect, indicating that ATP consumption is not due to opening of the mitochondrial permeability transition pore. We also wanted to determine if protein phosphorylation might be involved in regulating adenine nucleotide transport. To identify proteins that might be involved in the cardioprotective effect of GSKi, we performed western blot and 1D/2D gel phosphorylation site analysis using phos-tag staining to indicate proteins that had decreased phosphorylation in hearts treated with GSKi. LC/MS analysis revealed one of these proteins to be VDAC2. Taken together, we found that GSK mediated signaling may modulate transport through the outer membrane of the mitochondria. Both proteomics and adenine nucleotide transport data suggest the involvement of VDAC in GSK inhibited cardioprotection and suggest that VDAC is an important regulatory site in I/R injury.