Abstract 15891: Inhibition of Ceramide Synthesis by Myriocin Inhibits Cardiac Remodeling, Apoptosis and Proteolysis in Doxorubicin-Induced Cardiomyopathy
Background: Doxorubicin-induced apoptosis and cell death have been linked to increased ceramide synthesis. It is unclear whether doxorubicin-induced cardiomyopathy is associated with increased myocardial ceramides. We hypothesized that inhibition of de novo ceramide synthesis through myriocin inhibits pro-apoptotic pathways and proteolytic breakdown of structural and functional proteins in vitro and in vivo.
Methods: Cardiac dysfunction was induced in C57BL/6J mice (8 wks, n=40) by intraperitoneal injection of DOX (3mg/kg every other day for 2 wks) in the presence or absence of myriocin (0.3 mg/kg). Cardiac function was assessed by echocardiography. AC 16 cells were incubated with doxorubicin and C6 ceramide (DOX: 400 nM to 5 uM for 10 min to 16 hrs; C6 ceramide: 10uM) in the presence and absence of myriocin (10 uM). Cellular ceramides were analyzed by MC/MS lipidomics. Apoptosis was be assessed by DNA laddering and TUNEL assay. Proteolysis and ubiquitination were be assessed by ubiquitin-directed Co-IP followed by Western blotting.
Results: DOX decreased fractional shortening which was prevented by myriocin treatment (26.0±1.9 vs 33.0±1.3; p<0.01). Total cardiac ceramide content increased numerically in response to DOX (+25% vs +14%; p=NS) which was partially blocked by myriocin. In AC 16 cells, DOX increased ceramide content (+112.4%, p<0.01); especially C14 (+69%; p<0.05), C16 (+144%; p<0.01), C18 (+53%; p<0.05), C22 (+98%; p<0.05), C24 (+83.5%; p<0.01) and C24:1(+86%; p<0.05). Myriocin decreased very long-chain cellular ceramides C24:1 (-48%; p<0.05) and C22 (-47%; p<0.05) and reduced apoptosis. Myriocin partially blocked inactivation of pAKT and activation of pJNK induced by DOX which was induced by C6 ceramide (pAKT: -48%; p<0.01). Cardiac proteolysis and protein ubiquitination (TropI and MHC) under DOX was corrected by myriocin.
CONCLUSION: Cardiac ceramide content increases in DOX-associated cardiomyopathy and inhibition of de novo ceramide synthesis corrects cardiac ceramide content and composition. These findings provide a new understanding of lipotoxicity in DOX-associated cardiomyopathy and suggest ceramide synthesis modulation as a novel therapeutic option for the treatment of advanced heart failure.
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- © 2013 by American Heart Association, Inc.