Calcium-independent phospholipase A2 (iPLA2) is the predominant phospholipase A2 present in myocardium, and its pathophysiological role in acute myocardial infarction has been suggested by the rapid increase in membrane-associated iPLA2 activity during myocardial ischaemia and reperfusion (I/R). We therefore examined iPLA2 in mitochondrial fractions prepared from Langendorff-perfused adult rabbit hearts. Our studies indicate that iPLA2β is present in rabbit heart mitochondrial inner membranes with no apparent translocation during ischaemia, I/R or preconditioning. Mitochondrion-associated iPLA2 was catalytically competent and exhibited 2-, 3- and 2.5-fold increases in measured iPLA2 activity following ischaemia, I/R and preconditioning, respectively, when compared with the activity of iPLA2 measured in mitochondria from control hearts. Mitochondrial phospholipids are essential for maintaining the ordered structure and function of the organelle. I/R resulted in a rapid overall decrease in phosphatidylcholine and phosphatidylethanolamine glycerophospholipid species, as determined by electrospray ionization MS, that was partially alleviated by pretreatment of hearts with the iPLA2-specific inhibitor, bromoenol lactone (BEL). Pretreatment of I/R hearts with 10μM BEL significantly reduced the infarct size almost to that of continuously perfused hearts and was cardioprotective only when administered prior to ischaemia. Cardioprotection by BEL was reversed by the simultaneous perfusion of 100μM 5-hydroxydecanoate, implicating the mitochondrial KATP channel in BEL-mediated protection from I/R. Preconditioning also significantly reduced the infarct size in response to I/R but protection was lost by concurrent perfusion of 10μM arachidonic acid. Taken together, these data strongly implicate mitochondria-associated iPLA2 in the signal transduction of myocardial I/R injury.
Abbreviations used: iPLA2, calcium-independent phospholipase A2; cPLA2, cytosolic phospholipase A2; BEL, bromoenol lactone; ESI-MS, electrospray ionization MS; I/R, ischaemia and reperfusion; AACOCF3, arachidonyltrifluoromethyl ketone; BPE, 2-(4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-dodecanoyl)-1-hexadecanoyl-sn-glycero-3-phosphoethanolamine; TTC, 2,3,5-triphenyltetrazolium chloride; 5-HD, 5-hydroxydecanoic acid; EF, elongation factor.