Preconditioning decreases Bax expression, PMN accumulation and apoptosis in reperfused rat heart.
OBJECTIVE
Recent studies suggest that ischemic preconditioning (IPC) inhibits myocardial apoptosis after ischemia and reperfusion. This study tested the hypothesis that IPC reduces ischemia/reperfusion-induced myocardial apoptosis by inhibiting neutrophil (PMN) accumulation and altering expression of Bcl-2 and Bax proteins.


METHODS
Eighteen rats were subjected to 30 min of left coronary artery occlusion followed by 180 min of reperfusion with IPC (5 min ischemia and 10 min of reperfusion, n = 10) or without IPC (n = 8). Myocardial apoptosis was detected histologically using the terminal transferase UTP nick end labeling (TUNEL) assay and confirmed by DNA ladder on agarose gel electrophoresis. PMN accumulation was detected immunohistochemically with anti-rat CD18 antibody (WT3) and expression of Bcl-2 and Bax proteins was analyzed using Western blot assay.


RESULTS
IPC significantly decreased TUNEL positive cells (% total nuclei) in the ischemic zone from 28.6 +/- 2.8 to 3.4 +/- 0.9 (P < 0.05), consistent with the absence of DNA ladders in the IPC group. IPC significantly attenuated PMN accumulation (cells/mm2 myocardium) in the ischemic zone from 243 +/- 19 to 118 +/- 19 (P < 0.05). By regression analysis, there was a significant correlation between TUNEL positive cells and accumulated CD18 positive PMNs in the ischemic zone (r = 0.8, P < 0.001), which was shifted downward by IPC. Densitometrically, IPC significantly attenuated the ischemia/reperfusion-upregulated expression of Bax protein in the ischemic zone from 204 +/- 57% in the control group to 76 +/- 7% (P < 0.05), while the expression of Bcl-2 was not different from the non-ischemic zone in either group.


CONCLUSIONS
These data suggest that ischemic preconditioning may reduce myocardial apoptosis by inhibiting PMN accumulation and down-regulating expression of Bax.
Nakamura
M
M
Department of Cardiothoracic Surgery, Emory University School of Medicine, Atlanta, GA 30365-2225, USA.
Wang
N P
NP
Zhao
Z Q
ZQ
Wilcox
J N
JN
Thourani
V
V
Guyton
R A
RA
Vinten-Johansen
J
J
D000704:Analysis of Variance; D000818:Animals; D017209:Apoptosis; D004587:Electrophoresis, Agar Gel; D006321:Heart / Q000503:physiopathology*; D020287:In Situ Nick-End Labeling; D019157:Ischemic Preconditioning, Myocardial; D008297:Male; D015428:Myocardial Reperfusion Injury / Q000276:immunology / Q000378:metabolism / Q000503:physiopathology*; D020556:Neutrophil Infiltration; D011518:Proto-Oncogene Proteins / Q000378:metabolism*; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000378:metabolism; D051381:Rats; D051028:bcl-2-Associated X Protein
C494828:Bax protein, rat; D011518:Proto-Oncogene Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D051028:bcl-2-Associated X Protein