Antagonism of endothelin-1 inhibits hypoxia-induced apoptosis in cardiomyocytes.

Apoptosis is well documented to be a common feature of many pathological processes of the heart. Exogenous endothelin-1 (ET-1) has been shown to be proapoptotic or antiapoptotic, depending on ET-1 concentration, cell type, and the ratio of ETA/ETB receptor subtypes. The role of endogenous ET-1 in cardiomyocyte apoptosis, however, is not clarified. This study observed the effects of the ETA-receptor antagonists BQ610 and BQ123 and the ETB-receptor antagonist BQ788 on hypoxia-induced apoptosis in primary cultured neonatal rat cardiomyocytes. Hypoxic apoptosis was induced by incubating cardiomyocytes in serum-free medium under 3% O2 and 5% CO2 for 24 h and evaluated by TUNEL analysis and flow cytometry. TUNEL analysis showed that the apoptotic cardiomyocytes constituted 24.2% +/- 2.2% of the total cells under hypoxic conditions. Treatment with BQ610 (5 micromol/L) significantly reduced the apoptosis rate to 13.2% +/- 3.7% (data from 4 independent experiments, p < 0.01 vs. hypoxia). Flow cytometry showed that the percentage of apoptotic cells positively stained with annexin V and propidium iodide was 42.76% +/- 4.44% (n = 12) in cultures subjected to hypoxia. BQ123 at 0.04, 0.2, and 1.0 micromol/L dose-dependently reduced the apoptosis rate to 34.00% +/- 10.35% (n = 6, p < 0.05), 30.38% +/- 8.28% (n = 6, p < 0.01), and 22.89% +/- 4.19% (n = 6, p < 0.01), respectively. In contrast, BQ788 did not affect hypoxic apoptosis. These findings suggested that endogenous ET-1 contributed to hypoxia-induced apoptosis in cultured cardiomyocytes, which was mediated by ETA receptors, but not by ETB receptors.