Identification of the functional role of GSK3β in the ex vivo female rat heart at ischemic reperfusion--with special reference to Akt-dependent signalling
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Coronary heart disease is the leading cause of death worldwide, arising mainly from ischemic heart disease. Currently, the most effective method for reducing mortality in patients suffering from a coronary occlusion is rapid reperfusion of the ischemic myocardium. Paradoxically, reperfusion per se can also increase cell death, a phenomenon termed lethal reperfusion injury. The increased understanding of this mechanism, has led to novel research aiming to develop protective interventions against lethal reperfusion injury. Objective: The first aim of this study was to investigate whether treatment with insulin, the GSK3β-inhibitor SB-415286 or the combination of both, conferred cardioprotection in terms of reducing infarct size in the isolated ex vivo female heart, when administrated at the onset of ischemic-reperfusion. Secondly, the involvement of the prosurvival kinase Akt was explored to elucidate the intracellular signalling pathways involved in these cytoprotective treatments. Methods: The effect of the various treatments was assessed using the ex vivo isolated Langendorff perfused rat heart model. Female rat hearts underwent 20 minutes of stabilisation, 30 minutes of regional ischemia followed by 120 minutes of reperfusion ± insulin (0.3 mU/mL), GSK3β-inhibitor (3μM) or insulin+GSK3β-inhibitor treatment for 15 minutes at the onset of reperfusion. Measurements of infarct size as percentage of risk area were performed in order to evaluate the cardioprotective effect. To explore phosphorylation status of the prosurvival kinase Akt in response to the various treatments administrated at ischemic reperfusion, cardiac tissue was isolated and homogenised, followed by protein detection using SDS-PAGE and Western Blotting. Results: Administration of insulin, the GSK3β-inhibitor SB-415286 or the combination of the two, for 15 minutes from the onset of ischemic reperfusion, significantly reduced infarct size in the isolated ex vivo female rat heart as compared to control (Ins 29.9±5.5%, GSK3β-inh 29.4±2.7%, Ins+GSK3β-inh 27.1±5% vs. control 45.9±3.0%). No synergistic cardioprotective effect was observed with the combinational treatment, and also no difference in the response of female and male rat hearts was observed. Preliminary data suggest that phosphorylation of the prosurvival kinase Akt is involved in insulin mediated cardioprotection. However, its phosphorylation status is not affected by treatment with GSK3β-inhibitor. Conclusion: Insulin, the GSK3β-inhibitor SB-415286 and insulin+GSK3β-inhibitor confers cardioprotection in terms of reducing infarct size in the isolated ex vivo female rat heart, when administrated for 15 minutes at the immediate onset of reperfusion. Our preliminary data suggests that the prosurvival kinase Akt is involved in insulin treatment administered at reperfusion.
UtgiverThe University of Bergen
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