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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

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan (Y.-M.L., N.S., S.M., K.F.); Daiichi-Sankyo Pharmaceutical Co., Ltd. Tokyo, Japan (Y.S.); Institute of Pharmacology & Toxicology and Biochemical Pharmaceutics, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, China (F.H.); Department of Pharmacology, Soochow University School of Medicine, Suzhou, China (Z.-H.Q.); and Tohoku University 21st Century COE Program "CRESCENDO", Sendai, Japan (K.F.)

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 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-dimethoxyindazole (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 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 generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 µM) during the last 48 h inhibited the aberrant 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 attenuated by DY-9836 treatment. These results suggest that generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.

Address correspondence to: Dr. Kohji Fukunaga, Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Aramaki-Aoba Aoba-ku, Sendai 980-8578, Japan. E-mail: fukunaga{at}mail.pharm.tohoku.ac.jp