Ingrid Webster1, Angelique Smith1, Amanda Lochner2, Barbara Huisamen1,3. 1. Department of Biomedical Sciences, Division of Medical Physiology, Faculty of Medicine and Health Sciences, University of Stellenbosch, PO Box 19063, Tygerberg, 7505, South Africa. 2. Department of Biomedical Sciences, Division of Medical Physiology, Faculty of Medicine and Health Sciences, University of Stellenbosch, PO Box 19063, Tygerberg, 7505, South Africa. alo@sun.ac.za. 3. South African Medical Research Council, Tygerberg, South Africa.
Abstract
PURPOSE: The mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases that inactivate MAPKs by dephosphorylation. Impairment of MKP-1 expression in insulin resistance has been suggested to affect the cardioprotective properties of insulin. We hypothesized that manipulation of its activity during myocardial ischaemia/reperfusion of control as well as insulin-resistant rats may affect the outcome. METHODS: Hearts from 16 week dietary induced obese Wistar rats and their age matched controls were isolated, perfused in the working mode and subjected to 15 min global ischaemia / 30 min reperfusion or 35 min coronary artery ligation/ 60 min reperfusion. Hearts received insulin (1mIU/ml), a MKP-1 inhibitor (sanguinarine 2.5uM), or insulin + sanguinarine for 15 min pre- and 10 min post-ischaemia. Endpoints were functional recovery and infarct size. Hearts from control and experimental groups were freeze-clamped either immediately after removal from the animal (baseline) or at 10 min reperfusion after global ischaemia and Western blot analysis done for total and phosphorylated MKP-1. RESULTS: Insulin treatment significantly increased total work recovery while sanguinarine abolished the insulin-mediated protection. Insulin had no effect on infarct size while sanguinarine reduced infarct size. Insulin increased while sanguinarine attenuated phosphorylation of MKP-1 at 10 min reperfusion. CONCLUSION: Inhibition of MKP-1 with sanguinarine abolished the insulin-induced improvement in functional recovery, but reduced infarct size. Although the data suggest a role for this phosphatase in insulin-induced cardioprotection, the multiple downstream effects of insulin hamper interpretation of the data obtained. In addition, the effects of sanguinarine per se in myocardial ischaemia/reperfusion need to be further elucidated.
PURPOSE: The mitogen-activated protein kinase phosphatases (MKPs) are a family of dual-specificity phosphatases that inactivate MAPKs by dephosphorylation. Impairment of MKP-1 expression in insulin resistance has been suggested to affect the cardioprotective properties of insulin. We hypothesized that manipulation of its activity during myocardial ischaemia/reperfusion of control as well as insulin-resistant rats may affect the outcome. METHODS: Hearts from 16 week dietary induced obeseWistar rats and their age matched controls were isolated, perfused in the working mode and subjected to 15 min global ischaemia / 30 min reperfusion or 35 min coronary artery ligation/ 60 min reperfusion. Hearts received insulin (1mIU/ml), a MKP-1 inhibitor (sanguinarine 2.5uM), or insulin + sanguinarine for 15 min pre- and 10 min post-ischaemia. Endpoints were functional recovery and infarct size. Hearts from control and experimental groups were freeze-clamped either immediately after removal from the animal (baseline) or at 10 min reperfusion after global ischaemia and Western blot analysis done for total and phosphorylated MKP-1. RESULTS: Insulin treatment significantly increased total work recovery while sanguinarine abolished the insulin-mediated protection. Insulin had no effect on infarct size while sanguinarine reduced infarct size. Insulin increased while sanguinarine attenuated phosphorylation of MKP-1 at 10 min reperfusion. CONCLUSION: Inhibition of MKP-1 with sanguinarine abolished the insulin-induced improvement in functional recovery, but reduced infarct size. Although the data suggest a role for this phosphatase in insulin-induced cardioprotection, the multiple downstream effects of insulin hamper interpretation of the data obtained. In addition, the effects of sanguinarine per se in myocardial ischaemia/reperfusion need to be further elucidated.