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Am J Physiol Heart Circ Physiol (May 19, 2006). doi:10.1152/ajpheart.00798.2005
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Submitted on July 27, 2005
Accepted on April 11, 2006

Cardioprotection initiated by reactive oxygen species is dependent on the activation of PKC{epsilon}

Alamgir MN Kabir1, James E Clark, Masaya Tanno1, Xuebin Cao1, John S Hothershal2, Semjidnyam Dashnyam1, Diana A Gorog1, Mohamed Bellahcene1, Michael J Shattock1, and Michael S Marber1*

1 Department of Cardiology and Institute for Experimental Clinical Research, Skejby Hospital, Aarhus, Denmark
2 Medical Department, Aarhus Kommunehospital, Aarhus, Denmark

* To whom correspondence should be addressed. E-mail: mike.marber{at}kcl.ac.uk.

Objective: To examine whether cardioprotection initiated by reactive oxygen species (ROS) is dependent on PKC{epsilon}. Methods: Isolated buffer-perfused mouse hearts were randomised to 4 groups: 1. AntimycinA (AA) (0.1µg/ml) for 3min followed by 10min washout then 30min global ischaemia (I) and 2hours reperfusion (R). 2. Controls of I/R alone. 3. AA bracketed with 13min of N-2 Mercaptopropionyl glycine (MPG) followed by I/R. 4. MPG (200µM) alone followed by I/R. Isolated adult rat ventricular myocytes (ARVM) were exposed to AA (0.1µg/ml) and Lucigenin used to measure ROS production. Murine hearts and ARVM were exposed to AA (0.1µg/ml) with, or without, MPG and PKC{epsilon} translocation was measured by cell fractionation and subsequent western blot analysis. Finally, the dependence of AA protection on PKC{epsilon} was determined by the use of knockout mice (-/-) lacking PKC{epsilon}. Results: AA exposure caused ROS production which was abolished by the mitochondrial uncoupler FCCP. In addition, AA significantly reduced the percentage infarction/left ventricular volume compared with control I/R (26±4vs 43±2, p<0.05). Bracketing AA with MPG caused a loss of protection (52±7 vs 26±4, p<0.05). AA caused PKC{epsilon} translocation only in the absence of MPG and protection was lost on the pkc{epsilon}-/- background (38±3 vs 15±4 p<0.001). Conclusions: AA causes ROS production, on which protection and PKC{epsilon} translocation depend. In addition protection is absent in PKC{epsilon} null hearts. Our results imply that, in common with ischaemic preconditioning, PKC{epsilon} is crucial to ROS-mediated protection.




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