546 Cardiolipin - Protein Complexes and Initiation of Complement Activation After Coronary Artery Occlusion

Specific rabbit anti-cardiolipin (anti-CL) antibodies were used to investigate the hypothesis that cardiolipin, associated with mitochondrial membrane proteins, binds Cl and facilitates activation of the complement cascade following reperfusion of ischemic myocardium. By immunoelectron microscopy, anti-CL localized to subsarcolemmal mitochondria, emerging through breaks in membranes of damaged cardiac myocytes. Anti-CL reacted with >15 mitochondrial constituents, most of which comigrated with the proteins that bind Clq in transblots of subsarcolemmal mitochondria, fractionated by polyacrylamide gel electrophoresis under reducing conditions in the presence of sodium dodecyl sulfate. A subset of the Clq-binding proteins >24 to 37 kD served as stable sites for assembly of C3, C5, and C9. Cardiac lymph, collected during the first hour after reperfusion of ischemic myocardium, contained proteins of diverse size that reacted with both A ntibody-independent activation of the complement cascade has long been recognized as a consequence of myocardial ischemia.1-7 Clinical and laboratory investigations have demonstrated that activation of the complement cascade under these circumstances proceeds principally by the classic pathway.34,89 In experimental models of myocardial ischemia/reperfusion injury, one can show that cardiac extracellular fluids contain leukocyte chemotactic activity that is completely inhibited by antiserum to C5a.10 Release of this complement-dependent anaphylatoxin, after reperfusion of ischemic myocardium, activates circulating neutrophils and causes these to localize in regions of myocardium with significantly decreased blood flow.5"11 Complement-mediated inflammation accounts for a significant portion of ischemic injury, as shown by the myocardial salvage that results from treatments that suppress complement activation during ischemia and reperfusion.12"13 Although a number of studies have suggested that the proinflammatory stimulus can come from products of damaged cardiac myocytes,1-5 the initial trigger for this Received January 20, 1994; accepted May 20, 1994. From the Laboratories for Immunology (R.D.R.) and Syphilis (R.E.B.) Research at the Department of Veterans Affairs Medical Center; the Departments of Microbiology and Immunology (R.D.R., R.E.B.), Internal Medicine (R.D.R., L.H.M., K.Y., M.L.E.), Pediatrics (H.K.H., W.J.D.), and Pathology (H.K.H.), Baylor College of Medicine; and the Methodist Hospital (K.Y., M.L.E.), Houston, TX 77030. Correspondence to Roger D. Rossen, MD, Immunology Research Laboratory, Bldg 109, The VA Medical Center, 2002 Holcombe Blvd, Houston, TX 77030. ©) 1994 American Heart Association, Inc. anti-CL and Clq. Cardiac lymph, collected before occlusion and 4 to 5 hours after reperfusion, in comparison, had few if any Clq or anti-CL reactive proteins. Treatment with phospholipase suppressed the Clq-binding activity and anti-CL reactivity of the proteins in reperfusion lymph and those with similar properties in mitochondrial extracts. Our data suggest that during ischemia, mitochondria, extruded through breaks in the sarcolemma, unfold and release membrane fragments in which cardiolipin and protein are intimately associated. By binding Cl and supplying sites for the assembly of later-acting complement components, these fragments provide the means to disseminate the complement-mediated inflammatory response to ischemic injury. (Circ Res. 1994;75:546-555.)