Reperfusion induces myocardial apoptotic cell death.
OBJECTIVE
The purpose of the present study was to investigate whether apoptosis is triggered during ischemia (I) and reperfusion (R) and whether I/R-induced apoptosis is correlated with changes in expression of Bcl-2 and Bax.


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 electrophoresis or confirmed histologically using the terminal transferase UTP nick end labeling (TUNEL) assay.


RESULTS
Collateral myocardial coronary blood flow during I, confirmed by colored microspheres was comparable in both groups. Although PI caused 72 +/- 5% infarct size, very few TUNEL-positive cells were detected in the necrotic area (0.2 +/- 0.1% of total normal nuclei), consistent with an absence of DNA laddering. In contrast, the appearance of TUNEL-positive cells was significantly displayed after 6 h R in the necrotic area in I/R group (26 +/- 4%, P < 0.001 vs. PI group), and DNA ladder occurred in all experimental animals, suggesting that myocardial apoptosis is primarily elicited by R. Densitometrically, Western blot analysis showed significant reduction in expression of Bcl-2 (16 +/- 1%) and increase in Bax (29 +/- 8%) after 6 h R in the necrotic area compared with normal tissue while expression of these two proteins was not changed in the PI group. Polymorphonuclear neutrophil (PMN) accumulation in the necrotic area determined either by immunohistochemistry with anti-CD18 antibody or by myeloperoxidase activity was significantly increased in the I/R group compared to the PI group (358 +/- 24 vs. 24 +/- 2, mm2 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 relationship between CD18-positive PMNs and TUNEL-positive cells (P < 0.05) in the I/R group.


CONCLUSIONS
These results indicate that (1) PI without R did not induce apoptotic cell death, while two types of cell death, necrosis and apoptosis 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 development of apoptosis.
Zhao
Z Q
ZQ
Department of Surgery, Emory University School of Medicine, Atlanta, GA 30365-2225, USA.
Nakamura
M
M
Wang
N P
NP
Wilcox
J N
JN
Shearer
S
S
Ronson
R S
RS
Guyton
R A
RA
Vinten-Johansen
J
J
D000818:Animals; D017209:Apoptosis; D015153:Blotting, Western; D004285:Dogs; D005260:Female; D006321:Heart / Q000503:physiopathology*; D007150:Immunohistochemistry; D020287:In Situ Nick-End Labeling; D008297:Male; D017202:Myocardial Ischemia / Q000473:pathology / Q000503:physiopathology; D015428:Myocardial Reperfusion Injury / Q000473:pathology / Q000503:physiopathology*; D009504:Neutrophils / Q000473:pathology; D011518:Proto-Oncogene Proteins / Q000032:analysis; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000032:analysis; D051028:bcl-2-Associated X Protein
D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein