ORIGINAL CONTRIBUTION Basic Res Cardiol 95: 457 — 465 (2000) ' Steinkopff Verlag 2000 A. Gysembergh A. Zakaroff-Girard J. Loufoua L. Meunier X. Andr-Fout M. Lagarde A.-F. Prigent M. Ovize Brief preconditioning ischemia alters diacylglycerol content and composition in rabbit heart Abstract In order to give further insight into the potential role of PKC in the ben- ecial effects of ischemic preconditioning, we have characterized the production of diacylglycerol, the endogenous activator of PKC, and its molecular species compo- sition in ischemic control and preconditioned hearts. Preconditioning was induced by 1 cycle of 5 min of ischemia followed by 5 min of reperfusion. In control and preconditioned groups, hearts were harvested under deep anesthesia at baseline (preischemia) and at 2, 5 and 10 min into the sustained coronary artery occlusion, i.e., preceding myocyte death. Diacylglycerol content and fatty acid composition were analyzed by thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC), respectively. Myocardial diacylglycerol content was increased at 2 min into the sustained ischemia in the control group (481 – 34 vs 292 – 64 ng.mg —1 at baseline; p < 0.05), but was comparable to the baseline value at 5 and 10 min. In the preconditioned group, diacylglycerol production remained unchanged throughout the 10-min test ischemia (317 – 17 at 2 min vs 312 – 38 ng.mg —1 at baseline; p = NS). A detailed analysis of the molecular species composition at the time of 2 min revealed a reduced contribution of phosphatidy- linositol to diacylglycerol production in preconditioned myocardium (global corre- lation coefficient 0.57 vs 0.66 in control myocardium) with a trend toward an enrich- ment of diacylglycerol composition with some species originating from phos- phatidylcholine. Thus, our study revealed that brief preconditioning ischemia: (1) prevents the increase of diacylglycerol content in the early minutes of the sustained ischemia, and (2) emphasizes the contribution of phosphatidylcholine in diacyl- glycerol formation to the detriment of that of phosphatidylinositol. Key words Rabbit — preconditioning — diacylglycerol — molecular species — protein kinase C A. Gysembergh Æ J. Loufoua Æ M. Ovize Laboratoire de Physiologie Lyon-Nord Facult de Mdecine Lyon Nord Universit Claude Bernard Lyon I 8 Avenue Rockefeller 69373 Cedex 08, Lyon, France Prof. Michel Ovize () Æ L. Meunier X. Andr-Fout H opital Cardiovasculaire et Pneumologique Louis Pradel 59 Boulevard Pinel 69394 Cedex 03, Lyon, France e-mail: ovize@rockefeller1.univ-lyon1.fr A. Zakaroff-Girard Æ M. Lagarde A.-F. Prigent Inserm Unit 352 INSA-Lyon, B at. 406 20 Avenue A. Einstein 69621 Villeurbanne, France Received: 12 October 1999 Returned for 1. revision: 3 November 1999 1. Revision received: 6 January 2000 Returned for 2. revision: 14 February 2000 2. Revision received: 21 April 2000 Accepted: 9 May 2000 BRC 219 Introduction Preconditioning is a potent phenomenon whereby one or several brief episodes of ischemia can protect the heart from a subsequent episode of more sustained coronary artery occlu- sion (18). Evidence indicates that preconditioning the rabbit heart involves translocation and activation of protein kinase C (PKC), probably as a consequence of activation of G protein- coupled receptors (24, 35). This theory essentially relies on pharmacologic studies in which inhibitors of PKC were reported to abolish the protective effect of preconditioning (8, 27, 35, 37) whereas activators mimicked the benecial effects of an antecedent episode of ischemia. However, direct assessment of total PKC activity failed to conrm these obser- vations in the rabbit heart (30), albeit western immunoblotting experiments indicated an increase in sarcolemmal and nuclear