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 (PGI(2)) generation in cardiomyocytes. The siRNA transfection for PGI(2) synthase significantly reduced L-carnitine-induced PGI(2) and L-carnitine's protective effect. Furthermore, blockade the potential PGI(2) receptors, including PGI(2) 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 PGI(2) and PPARα-signaling pathways, which may potentially protect the heart from the severe toxicity of doxorubicin.
Chao
Hung-Hsin
HH
Department of Medicine, Taipei Medical University, Taipei, Taiwan, ROC.
Liu
Ju-Chi
JC
Hong
Hong-Jye
HJ
Lin
Jia-wei
JW
Chen
Cheng-Hsien
CH
Cheng
Tzu-Hurng
TH
D000818:Animals; D000831:Animals, Newborn; D000903:Antibiotics, Antineoplastic / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects*; D002331:Carnitine / Q000494:pharmacology*; D053148:Caspase 3 / Q000378:metabolism; D002478:Cells, Cultured; D045304:Cytochromes c / Q000378:metabolism; D004317:Doxorubicin / Q000633:toxicity; D011464:Epoprostenol / Q000378:metabolism*; D032383:Myocytes, Cardiac / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D019255:NADPH Oxidases / Q000378:metabolism; D047493:PPAR alpha / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D014803:Vitamin B Complex / Q000494:pharmacology*; D051020:bcl-X Protein / Q000378:metabolism
D000903:Antibiotics, Antineoplastic; C494813:Bcl2l1 protein, rat; D047493:PPAR alpha; D017382:Reactive Oxygen Species; D051020:bcl-X Protein; D014803:Vitamin B Complex; D004317:Doxorubicin; D045304:Cytochromes c; D011464:Epoprostenol; D019255:NADPH Oxidases; C505410:Casp3 protein, rat; D053148:Caspase 3; D002331:Carnitine