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Adenosine Attenuates Reperfusion-induced Apoptotic Cell Death by Modulating Expression of Bcl-2 and Bax Proteins☆

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Abstract

This study tests the hypothesis that infarct reduction with adenosine (Ado) is associated with inhibition of apoptotic cell death by modulating expression of anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins and reducing neutrophil accumulation. In three groups of dogs, the left anterior descending coronary artery was occluded for 60 min and reperfused for 6 h. Either saline (Control, n=8), Ado (140 μ g/kg/min, n=8) or CGS21680, an adenosine A2Areceptor analogue, (0.2 μ g/kg/min, n=7) were infused during the first 2 h of reperfusion. Myocardial apoptosis was detected by histological TUNEL staining and DNA laddering. Expression of Bcl-2 and Bax proteins was analyzed using Western blot assay. Neutrophil localization was detected by immunohistochemistry with monoclonal anti-neutrophil CD18 antibody. There was no group difference in collateral blood flow (colored microspheres) during ischemia. Intra-left atrial administration of Ado and CGS21680 significantly decreased infarct size from 26±2% in Control to 13±1%* and 16±3%*, respectively. TUNEL positive cells in the peri-necrotic zone of the ischemic myocardium were also significantly reduced from 16±2% in Control group to 9±1%* and 10±2%*, respectively, consistent with the absence of DNA laddering in these two groups. Densitometrically, Ado and CGS21680 at reperfusion significantly increased the expression (% of normal myocardium) of downregulated Bcl-2 from 45±6% in Control group to 78±12%* and 69±10%*, respectively, and attenuated expression of upregulated Bax from 198±16% in Control group to 148±10%* and 158±12%*, respectively. Furthermore, the number of positive CD18 cells (mm2myocardium), which was significantly correlated with TUNEL positive cells in peri-necrotic zone, was significantly reduced from 403±42 in Control group to 142±18* in Ado group and 153±20%* in CGS21680 group, respectively. In conclusion, the present study suggests that inhibition of apoptosis by Ado at reperfusion involves alterations in anti-apoptotic Bcl-2 and pro-apoptotic Bax proteins and neutrophil accumulation, primarily mediated by an adenosine A2Areceptor. * P<0.05 v Control group.

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    ☆

    Please address all correspondence to: Zhi-Qing Zhao, M.DPh.DThe Department of Cardiothoracic Surgery, The Carlyle Fraser Heart Center/Crawford Long Hospital, Emory University School of Medicine, 550 Peachtree St. N.E., Atlanta, GA 30365-2225, USA. E-mail: [email protected]

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