Brief Definitive Communication Protein Kinase C Its Role in Ischemic Preconditioning in the Rat

The present study investigated whether protein kinase C (PKC) plays a role in ischemic preconditioning in the rat heart. Chelerythrine, a specific antagonist of PKC, and 1,2-dioctanoyl-sn-glycerol (DOG), a diacylglycerol analogue and specific antagonist of PKC, were used to determine whether preconditioning could be blocked or triggered, respectively. Sprague-Dawley rats were anesthetized and instrumented for coronary occlusion and reperfusion. All animals were subjected to 45 minutes of regional ischemia (ISC) followed by 2.5 hours of reperfusion. The preconditioning protocol consisted of 5 minutes of ischemia and then 10 minutes of reperfusion. There were six groups: (1) control (group C, n=5), (2) preconditioned and ISC (group PC, n=6), (3) chelerythrine given 2 minutes before ISC (group CC, n=5), (4) preconditioned and chelerythrine given 2 minutes before ISC (group PCC, n=6), (5) DOG (dissolved in dimethylsulfoxide [DMSO]) given 10 minutes before ISC (group CD, n=5), and (6) DMSO given 10 minutes before ISC (group DMSO, n=3). The end point was infarct size measured using triphenyl tetrazolium chloride and expressed as a percentage of the volume at risk (I/R), measured with fluorescent partiI schemic preconditioning is the most powerful experimental strategy known for protecting the functioning heart from myocardial necrosis when it is subjected to ischemia.' The mechanism underlying preconditioning is unknown and is currently under intense investigation. There is evidence in support of adenosine being the most important endogenous mediator of protection in some animals (rabbits and dogs),1 where it is thought to act via the A1 receptor to trigger preconditioning. This response can be blocked or reproduced by giving an adenosine antagonist or agonist, respectively. However, adenosine does not appear to play the same role in the rat, and although Liu and Downey2 have induced protection in the rat by giving adenosine or an analogue of adenosine, this effect could not be blocked by giving an adenosine antagonist. Other investigators, such as Li and Kloner3 and Cave et al,4 have been unable to reproduce the effect of preconditioning in the rat with adenosine. This suggests that although adenosine may be involved in preconditioning in the rat, it is not the Received March 14, 1994; accepted May 19, 1994. From The Hatter Institute for Cardiovascular Studies, Division of Cardiology, University College London (England) Hospitals, and Victor Babes Institute (M.M.M.), Bucharest, Romania. Correspondence to Prof D.M. Yellon, The Hatter Institute for Cardiovascular Studies, Division of Academic Cardiology, University College London Hospitals, Grafton Way, London WC1E 6DB, England. © 1994 American Heart Association, Inc. cles. I/R was significantly reduced by preconditioning (group C 58.6+5.0%; group PC, 32.7±6.3%; P<.01) and by the PKC agonist DOG, which reduced I/R to a similar extent as preconditioning (group C, 58.6±5.0%; group CD, 28.0±7.0%; P<.01). Inhibition of PKC with chelerythrine abolished protection, there being no significant difference in I/R between control and the chelerythrine-treated preconditioned group (group C, 58.6±5.0%; group PCC, 62.3±5.7%). Chelerythrine and the vehicle DMSO had no direct influence on infarct size (group CC, 63.2+2.2%; group DMSO, 60.7±9.9%). Thus, PKC stimulation with the diacylglycerol analogue DOG resulted in protection, and PKC inhibition with chelerythrine abolished protection. The finding that treatment with a PKC stimulator gives a similar degree of protection against infarction as that seen after ischemic preconditioning and that this protection can be blocked by a PKC inhibitor provides support for the hypothesis that PKC plays an important role in ischemic preconditioning. (Circ Res. 1994;75:586-590.)