Progressively developed myocardial apoptotic cell death during late phase of reperfusion

Myocardial apoptosis is primarily triggered during reperfusion (R). The aim of this study was to test the hypothesis that R-induced apoptosis develops progressively during the late phase of R, and that R-induced apoptosis is associated with changes in expression of anti- and pro-apoptotic proteins a nd infiltrated inflammatory cells. Thirty-one dogs were subjected to 60 min of left anterior descending coronary occlusion followed by 6, 24, 48, and 72 h R, respectively. There was no group difference in collateral blood flo w, measured by colored microspheres during ischemia. Necrotic cell death (T TC staining) was significantly increased during R, starting at 27 +/- 2% at 6 h R and increasing to 41 +/- 2%dagger at 24 h R. There was no further ch ange at 48 (37 +/- 3%dagger) and 72 (36 +/- 6%dagger) h R, respectively. TU NEL positive cells (% total normal nuclei) in the peri-necrotic zone progre ssively increased from 6 (26 +/- 2*) to 24 (38 +/- 1*dagger), 48 (48 +/- 3* dagger) and 72 (59 +/- 4*dagger) h R, respectively. The number of detected TUNEL positive cells at these time points was consistent with an increased intensity of DNA ladders, identified by agarose gel electrophoresis. Compar ed with normal tissue, western blot analysis showed persistent reduction in expression of anti-apoptotic protein Bcl-2 from 6 (16 +/- 0.8%*) to 72 h R (78 +/- 2%*dagger), and increase in expression of pro-apoptotic proteins i ncluding Bax from 6 (30 +/- 3%*) to 72 h R (66 +/- 3%*dagger), and p53 from 6 (12 +/- 1%*) to 72 h R (91 +/- 2%*dagger), respectively. Immunohistochem ical staining revealed that infiltrated neutrophils (mm(2) myocardium) were significantly correlated with development of necrotic and apoptotic cell d eath from 6 to 24 h R, respectively (P < 0.05), while large macrophage infi ltration seen during 48 to 72 h R were correlated with apoptotic cell death (P < 0.05). These results indicate that 1) necrosis peaked at 24 h R when apoptosis was still progressively developing during later R; 2) changes in Bcl-2 family and p53 proteins may participate in R-induced myocardial apopt osis; 3) inflammatory cells may play a role in triggering cell death during R. *P < 0.05 vs. normal nuclei and tissue; daggerP < 0.01 vs. 6 h R.