Abstract 2326: The Effects of Ischemia-Reperfusion Injury on Bcl-2
Donor organ ischemia during heart transplantation increases myocardial apoptosis and chronic rejection, and this is coupled with a reduction of the anti-apoptotic protein, Bcl2. These findings suggest that donor organ apoptosis may play a key role in inducing immune activation and culminating in chronic rejection. We hypothesized that the ubiquitination regulates Bcl2 degradation in response to ischemia/reperfusion injury, initiating donor organ apoptosis Syngeneic heterotopic heart transplants were performed in Lewis rats with either 30, 60, or 90 minutes of total ischemia and were reperfused (I-R) for 4 hours and compared to native heart. Bcl2, Bax, and Cleaved Caspase 3 (CC3) were quantified with Western blots. Ubiquinated Bcl2 (Ubq-Bcl2) was quantified through Bcl2 immunoprecipitation and Ubiquitin Western blot. In vitro endothelial cells studies were performed with 6, 12, or 24 hours of ischemia followed by 6, 12 or 24 hours of reperfusion. A temporal profile of Bcl2, Bax, Ubq-Bcl2 and CC3 was created. In vivo: Donor organ ischemic time and serum Troponin I levels are directly correlated (R2 = 0.96). Bcl2 levels were 0.5 ± 0.08 and Bax levels were 2.2 ± 0.07 fold higher than native hearts after 90 minutes of ischemia (p < 0.05). There was a direct correlation between the Bax to Bcl2 ratio (Bax/Bcl2) and Troponin I levels (R2 = 0.94). Ubq-Bcl2 was 2.0 ± 0.04 fold higher than native hearts and peaked at 60 minutes of ischemia (p < 0.05). TUNEL positive cardiomyocytes increased with increasing ischemia (p < 0.05). In vitro: Bcl2 decreased 0.7 fold, Ubq-Bcl2 increased 2.3 fold, Bax remained constant, and CC3 increased 2.38 fold after 24 hours of ischemia compared to normoxic cells. With reperfusion, there was a reduction of Bcl2 and a peak of Ubq-Bcl2 after 12 hours of reperfusion at all ischemic times. Bax decreased by 0.2 fold after 6 hours of reperfusion at all ischemic times, but returned to baseline by 24 hours of reperfusion. However, similar to in vivo studies, the Bax/Bcl2 ratio increased. Cardiac injury after transplant is associated with decreased Bcl2 and increased Bax, and predominantly effects cardiomyocytes. Our data suggests that ubiquitination of Bcl2 may regulate its function during ischemia.