Proteasome Inhibitor Epoxomicin Prevents Cardiomyocyte Apoptosis through Regulation of Gene Expression

Abstract

Epoxomicin (E) is shown to prevent apoptosis during myocardial stress. In this study, we aimed to identify the molecular mechanism(s) associate with the anti‐apoptotic effect of E, using neonatal rat ventricular myocytes as a model system. The cultured cardiomyocytes were pretreated for 24 hrs with E. Apoptosis was induced by chelerythrine (Che) (5μM) for 35 min. Interestingly, Che induced apoptosis is associated with significant Akt phosphorylation at ser473. We found that E dose‐dependently (0.1μM) protects cardiomyocytes against apoptosis. Pretreatment of cardiomyocytes with E prevents Akt Ser473 phosphorylation and, thereby, apoptosis. The decrease in Akt Ser473 phosphorylation by E correlates with its protective effect in a time dependent manner. The phosphorylation of Akt at Thr308 was not altered by Che and/or by E treatment. To determine if the Akt Ser473 phosphorylation is proapoptotic, we ablated the expression of Rictor, a key component of mTORC2, which phosphorylates Akt at ser473. Rictor knockdown by RNA interference dramatically inhibits the Akt ser473 phosphorylation in Che treated cells, and reproduces the protective effects of E. Gene expression analysis shows that the expression of Akt substrate1 (Akt1S1), a negative regulator of mTORc kinase was significantly up‐regulated in E treated cells. These results together suggest that E inhibits the phosphorylation of Akt ser473 through negative regulation of Rictor activity. We further investigated the expression and phosphorylation patterns of upstream and downstream regulators of the Akt pathway. Our results show that Che activates the MEK/ERK signaling pathway and this activation was inhibited by E or by Rictor knockdown. In conclusions, our studies demonstrate that (1) the Akt activation, which is known to promote cell growth and survival, can also trigger cell death under certain conditions, and (2) in addition to the proteasome inhibition, E protects cardiomyocytes against apoptosis by changing gene expression.