Abstract 3303: The 26S Proteasome is Activated at Critically Low ATP Concentrations and Contributes to Myocardial Damage During Cold Ischemia
Proteasomes, the major non-lysosomal proteases in all eukaryotic cells, are regulated by ATP and thought to contribute to myocardial ischemia-reperfusion injury. However, their role and regulation during ischemia are poorly understood. Because the duration of cold ischemia (CI) is closely related to loss of function and reduced organ survival in cardiac transplantation, we studied whether proteasomes contribute to organ damage in rat hearts during CI (4°C). Consistent with earlier observations, we detected that proteasome activity in normal heart extracts is negatively regulated by ATP. Compared with normal myocardial [ATP, 5mM], proteasome activity increased continuously by 500% at 50–100 μM ATP when [ATP] was gradually reduced. Measurements of activities and 20S/26S proteasome content in highly purified enzyme preparations showed that this activation was caused by 26S proteasomes that are stable at low [ATP, <1mM]. Myocardial [ATP] during CI in University of Wisconsin (UW) solution for up to 48h decreased exponentially with a half life of 8.2h (r2: 0.8). Proteasome activities increased time dependently by 200% and reached plateau after 24h of CI when measured at the tissue [ATP] and 37°C, and by 1000% with plateau at 12h when measured at 4°C (p<0.001 vs. control for both conditions). Addition of the specific proteasome inhibitor epoxomicin (50 μM) to the UW solution reduced proteasome activities by >90%, but did not affect tissue [ATP]. While there were no histomorphological (H&E/Masson’s trichrome) differences between normal and ischemic hearts w/o epoxomicin, transmission electron microscopy showed profound ultrastructural changes after 24h of CI (cell edema, mitochondrial swelling, myofilament disruption), and all were obviously attenuated by epoxomicin. Wet/dry-weight ratios increased from 3.5±0.07 to 4.0±0.06 after 24h of CI (p<0.001) and were attenuated with epoxomicin (3.7±0.12, p<0.01 vs. CI; n=5/group). These findings identify the 26S proteasome as a protease that is activated at low tissue [ATP] and contributes to myocardial damage during CI. Our data further suggest proteasome inhibition as a novel approach to reduce cell damage during cold ischemic storage of donor organs, and possibly during myocardial ischemia in general.
This research has received full or partial funding support from the American Heart Association, Greater Southeast Affiliate (Alabama, Florida, Georgia, Louisiana, Mississippi, Puerto Rico & Tennessee).