Abstract 215: Arc Transcriptional Regulation And Degradation Is A Critical Cardiomyocyte Survival Switch In Doxorubicin-cardiotoxicity
Despite its complexity of action doxorubicin (Dox)-induced cardiomyopathy results in loss of cardiomyocytes which further contributes to the development of overt heart failure. We showed recently that the apoptosis-repressor-with-CARD (ARC) protects cardiomyocytes from biomechanical and ischemic stress. In the present study, we examined the relevance of ARC on cardiomyocyte survival and its underlying mechanisms in a model of Dox-induced cardiotoxicity. Exposure of neonatal rat ventricular cardiomyocytes with Dox (1μM) resulted in a time-and dose-dependent downregulation of ARC that was associated with an upregulation of ARC repressor p53 and ubiquitin E3 ligase MDM2. Dox exposure led to a reduction of ARC mRNA levels (RT-PCR, n=5, p<0.05 vs control) indicating transcriptional repression and showed a substantial induction of apoptosis (trypan blue exclusion, n=5, p<0.01 vs control). Proteasomal inhibitors MG132 and lactacystin partially rescued both Dox-induced downregulation of ARC and induction of apoptosis. In contrast, enforced expression of ARC by adenoviral-mediated gene transfer dramatically increased the resistance of myocytes to undergo apoptosis following Dox (trypan blue exclusion, n=5, p<0.05 vs AdβGal+Dox; cell death ELISA, n=4, p<0.01 vs AdβGal+Dox). In response to Dox Bax translocated from cytosol to mitochondria where it resulted in cytochrome c release and dissipation of the mitochondrial membrane potential. Release of cytochrome c was associated with a 3-fold increase in caspase-9/-3 catalytic activity but independent of Bid cleavage and caspase-8 activation. ARC prevented Bax translocation to the mitochondrium and thereby blocked the activation of the mitochondrial apoptotic pathway. We further tested the regulation of endogenous ARC in a mouse model of acute Dox toxicity. Similar to our in vitro results Dox stimulation led to a downregulation of ARC that was associated with elevated levels of p53 and MDM2 protein. In conclusion we provide evidence for the protective role of antiapoptotic ARC in Dox-induced cardiotoxicity. Our data suggest that ARC repression and degradation represent critical steps in the development of Dox-induced myocardial damage which makes this molecule a target for future therapies.