Abstract 851: The Cardioprotective Effect Of Glycogen Synthase Kinase-3β (gsk-3β) Inhibitors Involves Inhibition Of Mitochondrial Adenine Nucleotide Transport
Inhibition of GSK-3β has been shown to reduce ischemia-reperfusion injury by acting on the mitochondria. We aimed to determine the mechanism by which inhibition of GSK-3β mediates cardioprotection. Rats were treated with specific GSK-3β inhibitors, SB216763 (3 μM) or SB415286 (10 μM) 15 min prior to ischemia. Both inhibitors significantly improved post-ischemic left ventricular function after 20 minutes of ischemia compared to control. Mitochondria were isolated from all three groups of hearts immediately after 15 min perfusion with/without GSK-3β inhibitors. Both the SB compounds significantly reduced state 3 respiration as well as ATP consumption during anoxia. Consumption of ATP during anoxia was measured by allowing mitochondria in an oxygraph chamber to consume oxygen until they became anoxic, and the rate of consumption of added ATP was measured at 20, 40, and 60 minutes of anoxia. GSK-3β inhibitors significantly slowed anoxic mitochondrial ATP consumption. Similarly, GSK-3β inhibitors significantly reduced ATP consumption when sodium cyanide was used to stop mitochondrial respiration and also when the mitochondria were deenergized by uncoupler addition. This reduction in ATP consumption could be due to inhibition of ATP entry into the mitochondria through Voltage dependent anion channel (VDAC) and/or the adenine nucleotide transporter (ANT), inhibition of some other unknown protein which may directly inhibit mitochondrial transport, or inhibition of the F1F0 ATPase. To identify proteins that might be involved in this reduced ATP consumption, western blot analysis with phospho-Akt substrate antibody and 1-D gel phosphorylation site analysis staining revealed the GSK-3β inhibitors significantly decrease the phosphorylation of a 32 kDa protein compared to control mitochondria. We then cut out the 32 kDa band and used mass spectrometry to identify proteins. Voltage dependent anion channel members 1, 2 and 3 were all identified along with the adenine nucleotide translocator and γ-subunit of the F1 ATPase. Taken together the data indicate that GSK-3β regulates mitochondrial energetics under anoxic conditions, which may play an important role in cardioprotection.