Apoptosis in rat cardiac myocytes induced by Fas ligand: Priming for Fas-mediated apoptosis with doxorubicin

Fas/Fas ligand (FasL) is well known for its role in delivering apoptotic signals; however, it is unclear whether Fast can mediate apoptosis in cardiomyocytes, We hypothesized that apoptosis via Fas/FasL system may be augmented in damaged cardiomyocytes. To determine whether Fast mediates cardiomyocyte apoptosis, recombinant Fast (rFasL) was added to the culture of neonatal rat ventricular myocytes pretreated with and without: doxorubicin. Without doxorubicin, high dose of rFasL caused an increase in TUNEL-positive cardiomyocytes and a mild decrease in MTT activities. When cardiomyocytes were pretreated with doxorubicin (0.5 mu M), rFasL dramatically augmented TUNEL-positive cardiomyocytes in a concentration-dependent manner, which was accompanied with nuclear fragmentations. The rFasL, also caused a concentration-dependent reduction in MTT activities in cardiomyocytes. The rFasL-induced caspase-8 activity was greatly facilitated by pretreatment of doxorubicin. TUNEL-positive nuclei with rFasL was inhibited by FasFc, neutralizing agent of rFasL, and Z-IETD-FMK, caspase-8 inhibitor. Fas mRNA transcript by RT-PCR was upregulated in cardiomyocytes with doxorubicin. We conclude that Fast can induce cardiomyocyte apoptosis particularly when cardiomyocyte becomes susceptible for Fas-mediated apoptosis. (C) 2000 Academic Press.