Abstract 17320: Inhibition of Ceramide Synthesis Preserves Cardiac Function and Increases Survival in Doxorubicin-induced Cardiomyopathy

Abstract

Background: Previous studies have linked increased ceramide and dihydroceramide (DHC) synthesis to cardiotoxicity. However, the effects of ceramide synthesis inhibition on doxorubicin (DOX)-induced cardiomyopathy are unclear. Hypothesis: Inhibition of de novo ceramide synthesis by myriocin maintains heart function and survival rate. Methods: C57BL/6J mice (8 wks, n=52) were treated with DOX (3 mg/kg every other day for 2 wks) with or without myriocin (0.3 mg/kg). Cardiac function was assessed by echocardiography. AC16 cardiomyocyte-like cells were incubated with DOX in the presence or absence of myriocin (10 uM). Ceramides were analyzed by LC/MS lipidomics. Reactive oxygen species (ROS) were measured by FACS (CellROX) and mitochondrial function determined by Seahorse analyzer. Results: In DOX-treated mice, inhibition of ceramide synthesis by myriocin preserved cardiac fractional shortening (29.2±2.7 vs 36.9±1.4% in myriocin; p<0.05), left ventricular systolic diameter dilation (3.2±0.1 vs 2.97±0.8 mm, p<0.05), running endurance (8.5 vs 17.5 min; p<0.05) and survival rates (35 vs 90% in myriocin, p<0.001). DOX increased cardiac total ceramides (+70%; p<0.001) and DHC (+73%; p<0.05); these increases were prevented by myriocin. Cardiac apoptosis in these cells was increased 4.1 fold by DOX and reduced by 50% with myriocin (p<0.05). In cells, DOX increased cellular 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). Interestingly, ceramide accumulation in mitochondria of DOX-treated cells (+125%; p<0.05) was associated with decreased mitochondrial respiration (-76%; p<0.05) and respiratory capacity (-88%; p<0.05). Myriocin treatment decreased very long-chain ceramides (-52%; p<0.05), reduced apoptosis (-23%; p<0.01), and prevented proteolysis of actin protein ubiquitination (-31%; p<0.05). Also, myriocin inhibited DOX-induced increase of ROS (+32%; p<0.05). Conclusion: Cardiomyocyte ceramide accumulation, especially in mitochondria, is associated with mitochondrial dysfunction, cardiotoxicity and reduced survival. Pharmacological inhibition of ceramide synthesis may prevent lipotoxicity.