Association of increased ubiquitinated proteins with cardiac apoptosis

Intracellular proteases play an important role in the regulation of apoptosis. A study was performed to determine whether inhibition of the cardiac ATP-dependent ubiquitin 26S protease complex affects cardiomyocyte apoptosis. Isolated rat hearts were perfused for up to 80 min with Krebs-Henseleit buffer +/- the 26S-proteasome inhibitor, MG132 (Z-leu-leu-leucinal). TUNEL-staining of hearts perfused with 25 microM MG132 for 50 min revealed a significant increase (p < 0.05) in the apoptotic index from 1.1% to 15.5% when compared with control hearts perfused with buffer only. Histology of adjacent myocardial sections revealed no signs of necrotic or late apoptotic (nuclear condensation) changes, indicating that the TUNEL-positive nuclei were in the early stages of apoptosis. This early stage of apoptosis was associated with a significant (p < 0.05) reduction in cardiac function. There was a 63% decrease in the rate pressure product in hearts perfused with 25 microM MG132 as compared with a 35% decrease in control hearts over the 80-min perfusion period. Soluble ubiquitin-conjugated proteins, as detected by probing with a specific antibody to ubiquitin, were increased in MG132-treated hearts. In hearts perfused with 50 microM MG132, a greater accumulation of ubiquinated proteins was observed accompanied by a more rapid and greater reduction in hemodynamic function. These observations indicate that prolonged inhibition of the ubiquitin-26S-proteasome results in cardiomyocyte apoptosis accompanied by increased ubiquinated proteins, thus suggesting that accumulation of these abnormal proteins may act as a signal to activate the cell death program.