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(Circulation. 2001;104:I-202.)
© 2001 American Heart Association, Inc.

Thoracic Transplantation and Ventricular Assist Devices

Upregulation of Bcl-2 Through Caspase-3 Inhibition Ameliorates Ischemia/Reperfusion Injury in Rat Cardiac Allografts

Jürg Grünenfelder, MD; Douglas N. Miniati, MD; Seiichiro Murata, MD; Volkmar Falk, MD; E. Grant Hoyt; Murray Kown, MD; Mark L. Koransky, MD; Robert C. Robbins, MD

Department of Cardiothoracic Surgery, Stanford University School of Medicine, Stanford, Calif.

Correspondence to Robert C. Robbins, Department of Cardiothoracic Surgery, Falk Cardiovascular Research Bldg, Stanford University Medical Center, Stanford, CA 94305. E-mail robbins{at}stanford.edu

Background— Oxidative stress after ischemia/reperfusion of cardiac allografts leads to cytokine production. Bcl-2, an inhibitor of apoptosis, also has strong antioxidant properties. Caspase-3 is known to cleave bcl-2. This study tests the hypothesis that bcl-2 is downregulated while tumor necrosis factor- (TNF-) levels increase after cardiac transplantation. Furthermore, the use of caspase-3 inhibition was investigated as a strategy for preserving myocardial bcl-2 and mitochondrial cytochrome c after transplantation.

Methods and Results— PVG-to-ACI rat heterotopic cardiac transplantations were performed in 4 groups designed with 30 minutes’ ischemia and 4 or 8 hours of reperfusion (n=4 per group). Treatment consisted of DEVD-CHO 500 µg IP per animal to donor and recipient 2 hours before transplantation and 250 µg IC into allograft. Controls were treated with saline. Grafts were analyzed by reverse transcription–polymerase chain reaction for bcl-2 mRNA, by ELISA for TNF-, for myeloperoxidase activity, and by Western blot for cytochrome c. In untreated groups, bcl-2 mRNA decreased significantly over time, whereas TNF- increased significantly at 4 hours (P=0.003) and returned to baseline after 8 hours’ reperfusion (P=NS compared with normal hearts). Treatment with caspase-3 inhibitor showed significant upregulation of bcl-2 mRNA expression after 4 and 8 hours of reperfusion (P<0.001 versus control), with a concomitant decrease in TNF- to baseline levels. Myeloperoxidase activity in all groups was no different from that of normal hearts. Mitochondrial cytochrome c release increased in both control and treatment groups.

Conclusions— Bcl-2 is actively downregulated and TNF- is upregulated in this model of cardiac allograft ischemia/reperfusion. Furthermore, the caspase-3 pathway is linked to this process, and blockade of caspase-3 can ameliorate reperfusion injury by upregulating bcl-2 and inhibiting TNF- without affecting cytochrome c release.

Key Words: ischemia • transplantation • reperfusion • inhibitors