L-carnitine reduces doxorubicin-induced apoptosis through a prostacyclin-mediated pathway in neonatal rat cardiomyocytes

Background: Clinical use of doxorubicin is greatly limited by its severe cardiotoxic side effects. L-carnitine is a vitamin-like substance which has been successfully used in many cardiomyopathies, however, the intracellular mechanism(s) remain unclear. The objective of this study was set to evaluate the protective effect of L-carnitine on doxorubicin-induced cardiomyocyte apoptosis, and to explore its intracellular mechanism(s). Methods: Primary cultured neonatal rat cardiomyocytes were treated with doxorubicin (1 μM) with or without pretreatment with L-carnitine (1-30 mM). Lactate dehydrogenase assay, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling staining, and flow cytometry measurement were used to assess cytotoxicity and apoptosis. Fluorescent probes 2',7'-dichlorofluorescein diacetate and chemiluminescence assay of superoxide production were used to detect the production of reactive oxygen species. Western blotting was used to evaluate the quantity of cleaved caspase-3, cytosol cytochrome c, and Bcl-x _L expression. Results: L-carnitine inhibited doxorubicin-induced reactive oxygen species generation and NADPH oxidase activation, reduced the quantity of cleaved caspase-3 and cytosol cytochrome c, and increased Bcl-x _L expression, resulting in protecting cardiomyocytes from doxorubicin-induced apoptosis. In addition, L-carnitine was found to increase the prostacyclin (PGI₂) generation in cardiomyocytes. The siRNA transfection for PGI₂ synthase significantly reduced L-carnitine-induced PGI₂ and L-carnitine's protective effect. Furthermore, blockade the potential PGI₂ receptors, including PGI₂ receptors (IP receptors), and peroxisome proliferator-activated receptors alpha and delta (PPARα and PPARδ), revealed that the siRNA-mediated blockage of PPARα considerably reduced the anti-apoptotic effect of L-carnitine. Conclusions: These findings suggest that L-carnitine protects cardiomyocytes from doxorubicin-induced apoptosis in part through PGI₂ and PPARα-signaling pathways, which may potentially protect the heart from the severe toxicity of doxorubicin.