Phenylephrine-Induced Cardiomyocyte Injury Is Triggered by Superoxide Generation through Uncoupled Endothelial Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836), a Novel Calmodulin Antagonist

The pathophysiological relevance of endothelial nitric-oxide synthase (eNOS)-induced superoxide production in cardiomyocyte injury after prolonged phenylephrine (PE) exposure remains unclear. The aims of this study were to define the mechanism of O-2(radical anion) 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 O-2(radical anion) production were monitored in PE-treated cells by 4-amino-5-methylamino-2',7'-difluorofluorescein 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 O-2(radical anion) generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 mu M) during the last 48 h inhibited the aberrant O-2(radical anion) generation nearly completely and NO production by 72% (p < 0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TdT-mediated dUTP nick-end labeling staining was also attenuated by DY-9836 treatment. These results suggest that O-2(radical anion) generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O-2(radical anion) and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.