Investigating and re-evaluating the role of GSK3beta kinase as a molecular target for cardioprotection by using novel pharmacological inhibitors.

Aims Glycogen synthase kinase 3 beta ( GSK3 beta) link with the mitochondrial Permeability Transition Pore ( mPTP) in cardioprotection is debated. We investigated the role of GSK3 beta in ischaemia ( I)/reperfusion ( R) injury using pharmacological tools. Methods and results Infarct size using the GSK3 beta inhibitor BIO ( 6-bromoindirubin-30-oxime) and several novel analogues ( MLS2776-MLS2779) was determined in anaesthetized rabbits and mice. In myocardial tissue GSK3 beta inhibition and the specificity of the compounds was tested. The mechanism of protection focused on autophagy-related proteins. GSK3 beta localization was determined in subsarcolemmal ( SSM) and interfibrillar mitochondria ( IFM) isolated from Langendorff-perfused murine hearts ( 30' I/10' R or normoxic conditions). Calcium retention capacity ( CRC) was determined in mitochondria after administration of the inhibitors in mice and in vitro. The effects of the inhibitors on mitochondrial respiration, reactive oxygen species ( ROS) formation, ATP production, or hydrolysis were measured in SSM at baseline. Cyclosporine A ( CsA) was co-administered with the inhibitors to address putative additive cardioprotective effects. Rabbits and mice treated with MLS compounds had smaller infarct size compared with control. In rabbits, MLS2776 and MLS2778 possessed greater infarct-sparing effects than BIO. GSK3 beta inhibition was confirmed at the 10th min and 2 h of reperfusion, while up-regulation of autophagy-related proteins was evident at late reperfusion. The mitochondrial amount of GSK3 beta was similar in normoxic SSM and IFM and was not altered by I/R. The inhibitors did not affect CRC or respiration, ROS and ATP production/hydrolysis at baseline. The co-administration of CsA ensured that cardioprotection was CypD-independent. Conclusion Pharmacological inhibition of GSK3 beta attenuates infarct size beyond mPTP inhibition.