Reperfusion induces myocardial apoptotic cell death

Objective: The purpose of the present study was to investigate whether apop tosis is triggered during ischemia (I) and reperfusion (R) and whether I/R- induced apoptosis is correlated with changes in expression of Bcl-2 and Pax . Methods: Anesthetized open-chest dogs were divided into two groups. Group I: 7 h of permanent I without R (PI, n=7); Group II: 60 min I followed by 6 h R (I/R, n=8). Apoptosis was identified as "DNA ladder" by agarose gel e lectrophoresis or confirmed histologically using the terminal transferase U TP nick end labeling (TUNEL) assay. Results: Collateral myocardial coronary blood flow during I, confirmed by colored microspheres was comparable in b oth groups. Although PI caused 72+/-5% infarct size, very few TUNEL-positiv e cells were detected in the necrotic area (0.2+/-0.1% of total normal nucl ei), consistent with an absence of DNA laddering. In contrast, the appearan ce of TUNEL-positive cells was significantly displayed after 6 h R in the n ecrotic area in I/R group (26+/-4%, P<0.001 vs. PI group), and DNA ladder o ccurred in all experimental animals, suggesting that myocardial apoptosis i s primarily elicited by R. Densitometrically, Western blot analysis showed significant reduction in expression of Bcl-2 (16+/-1%) and increase in Pax (29+/-8%) after 6 h R in the necrotic area compared with normal tissue whil e expression of these two proteins was not changed in the PI group. Polymor phonuclear neutrophil (PMN) accumulation in the necrotic area determined ei ther by immunohistochemistry with anti-CD18 antibody or by myeloperoxidase activity was significantly increased in the I/R group compared to the PI gr oup (358+/-24 vs. 24+/-2, mm(2) myocardium, P<0.01) and (2.9+/-0.3 vs. 0.4/-0.1, U/100 mg tissue, P<0.01). There was a significant linear relationshi p between CD18-positive PMNs and TUNEL-positive cells (P<0.05) in the I/R g roup. Conclusions: These results indicate that (I) PI without R did not ind uce apoptotic cell death, while two types of cell death, necrosis and apopt osis were found after I/R, (2) the Bcl-2 family may participate in early R- induced myocardial apoptosis, (3) PMN accumulation may play a role in the d evelopment of apoptosis. (C) 2000 Elsevier Science B.V. All rights reserved .