The pathophysiological relevance of endothelial nitric-oxide synthase (eNOS)-induced superoxide production in cardiomyo- cyte injury after prolonged phenylephrine (PE) exposure remains unclear. The aims of this study were to define the mechanism of O2- production by uncoupled eNOS and evaluate the therapeutic potential of a novel calmodulin antagonist 3-[2-[4- (3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5, 6-dimethoxy- indazole (DY-9836) to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96 h to PE led to translocation from membrane to cytosol of eNOS and. breakdown of caveolin-3 and dystrophin. When NO and O2- production were monitored in PE-treated cells by 4-amino-5-methylamino-2′,7′-difluorofluo- rescein and dihydroethidium, respectively, Ca2+-induced NO production elevated by 5.7-fold (p < 0.01) after 48-h PE treatment, and the basal NO concentration markedly elevated (16-fold; p < 0.01) after 96-h PE treatment. On the other hand, the O2- generation at 96 h was closely associated with an increased uncoupled eNOS level. C]oincubation wi.th DY-9836 (3 μM) during the last 48 h inhibited the aberrant O2- generation nearly completely and NO production by 72% (p < 0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin- 3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TdT-mediated dUTP nick-end labeling staining was also att.enuated by DY-9836 treatment. These results suggest that O2- generation by uncoupled eNOS probably triggers PE- induced cardiomyocyte injury. Inhibition of abnormal O 2- and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.