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.
Department of Obstetrics and Gynecology, Winthrop University Hospital, Mineola, New York 11501, USA. Spowell@Winthrop.org
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Powell
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Gurzenda
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Teichberg
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Mantell
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Maulik
D000818:Animals; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D003546:Cysteine Endopeptidases; D006339:Heart Rate / Q000187:drug effects; D006439:Hemodynamics / Q000187:drug effects; D020287:In Situ Nick-End Labeling; D007976:Leupeptins / Q000494:pharmacology; D008297:Male; D009097:Multienzyme Complexes / Q000037:antagonists & inhibitors; D009124:Muscle Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D009200:Myocardial Contraction / Q000187:drug effects; D009206:Myocardium / Q000166:cytology*; D011480:Protease Inhibitors / Q000494:pharmacology; D046988:Proteasome Endopeptidase Complex; D011499:Protein Processing, Post-Translational / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley; D014452:Ubiquitins / Q000378:metabolism*
D007976:Leupeptins; D009097:Multienzyme Complexes; D009124:Muscle Proteins; D011480:Protease Inhibitors; D014452:Ubiquitins; D003546:Cysteine Endopeptidases; D046988:Proteasome Endopeptidase Complex; C072553:benzyloxycarbonylleucyl-leucyl-leucine aldehyde