Characterisation of Temperature Preconditioning of Adult Rat Ventricular Myocytes
thesisposted on 04.10.2013, 10:03 by Yusuf Bhagatte
Temperature preconditioning is a relatively novel cardioprotective intervention, demonstrated to protect ex vivo isolated rat hearts against ischaemia- reperfusion injury. For the first time, the effect of temperature preconditioning on isolated ventricular myocytes was investigated in this study. This was followed by characterisation of the molecular mechanisms involved in temperature preconditioning. Temperature preconditioning (16°C) was found to be cardioprotective in isolated adult rat ventricular myocytes enhancing contractile recovery and preventing calcium dysregulation after metabolic inhibition and re-energisation (simulated ischaemia-reperfusion). Temperature preconditioning also preserved mitochondrial function by delaying the pathological opening of the mitochondrial permeability transition pore (mPTP) in a model of reperfusion injury. For the first time, reactive oxygen species (ROS) are shown to be released from the mitochondria exclusively during the hypothermic episodes of the temperature preconditioning protocol. This was characterised using a mitochondrially targeted ROS biosensor and ROS release was observed during the brief bursts to 16°C during temperature preconditioning. A ROS scavenger (MPG) significantly attenuated ROS accumulation during temperature preconditioning and consequently abolished the temperature preconditioning-induced protective delay in mPTP opening. Western blot analysis revealed temperature preconditioning phosphorylation of the pro-survival kinase ERK1/2. ERK1/2 activation was shown to be downstream of ROS release as the presence of a ROS scavenger during temperature preconditioning completely blocked ERK1/2 activation. The cardioprotective effects of temperature preconditioning on mPTP opening were completely lost by inhibiting ERK1/2 activation. Thus mitochondrial ROS release and ERK1/2 activation are both necessary to signal the cardioprotective effects of temperature preconditioning in cardiac myocytes.