Received for publication July 18, 2008.
Revised October 24, 2008.
Accepted for publication October 24, 2008.

Phenylephrine-induced cardiomyocyte injury is triggered by superoxide generation through uncoupled eNOS and ameliorated by DY9836, a novel calmodulin antagonist

Abstract

The pathophysiological relevance of eNOS-induced superoxide production in cardiomyocyte injury following prolonged 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, DY-9836, {3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole}, to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96h 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 DAF-FM and DHE, 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. Co-incubation with DY-9836 (3 µM) during the last 48 h inhibited the aberrant O₂^.- generation near 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 TUNEL staining was also attenuated by DY-9836 treatment. These results suggest that O₂^.- generation by uncoupled eNOS likely triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O₂^.- and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.

Key words: Dopamine, Glutamate, Nitric oxide, Nitric oxide synthases, Apoptosis, Oxidative stress/antioxidants