Doxorubicin induces slow ceramide accumulation and late apoptosis in cultured adult rat ventricular myocytes

Objectives: Anthracyclines cause apoptotic death in many cell types through activation of the ceramide pathway. We tested the hypothesis that doxorubi cin induces cardiac myocyte apoptosis through ceramide generation. Methods: Adult rat ventricular myocytes were grown in the presence of 10% fetal cal f serum, and exposed to 0.5 mu M doxorubicin (Dox) for 1 h on the day of ce ll isolation (day 0). We used the membrane-permeant ceramide analog C-2-cer amide (C-2-cer) to mimic the effects of endogenous ceramide and PDMP to ind uce endogenous ceramide accumulation. Apoptosis was assessed upon morpholog ical criteria and DNA fragmentation by the TUNEL method and agarose gel ele ctrophoresis. Ceramide concentration was assessed using the DAG kinase assa y. Results: Myocyte exposure to Dox was associated with cellular and nuclea r alterations typical of apoptosis on day 7 but not on day 3. At day 7, the percentage of TUNEL-positive myocytes was markedly increased in Dox-treate d cultures compared to control (Cl) cultures (82+/-3 vs. 12+/-1%, n=7; p<0. 001); internucleosomal DNA fragmentation was confirmed by the observation o f DNA ladders. These alterations were associated with an increase in the in tracellular ceramide concentration (1715+/-243 vs. 785+/-99 pmol/mg prot, n =5; p<0.01), a phenomenon also detected on day 3 (731+/-59 vs. 259+/-37 pmo l/mg prot, n=5; p<0.001). Incubation of myocytes at day 0 with 50 mu M C-2- cer induced rapid cell shrinkage and DNA fragmentation (45+/-3 vs. 10+/-1% TUNEL-positive myocytes on day 1 in C-2-cer-treated and Cl cultures, respec tively; n=6, p<0.001). Myocyte exposure to 10 mu M PDMP for 7 days (n=5), c aused ceramide accumulation (1.7-fold increase vs. Cl, p<0.01), and a marke d increase in the percentage of TUNEL-positive myocytes (62+/-6 vs. 11+/-3% in Cl cultures, p<0.001). Ventricles of rats injected intraperitoneally wi th a cumulative dose of 14 mg/kg Dox over a period of 2 weeks also showed a n increased ceramide concentration 2 weeks later (11.01+/-0.64 vs. 5.24+/-0 .88 pmol/mg prot, n=6; p<0.001). Conclusion: Our study confirms the existen ce of a functional ceramide pathway related to apoptosis in cardiac myocyte s, and points to its possible involvement in doxorubicin-induced cardiomyop athy. (C) 1999 Published by Elsevier Science B.V. All rights reserved.