Aims: 

Reactive oxygen species (ROS) are produced during normal aerobic metabolism in the heart. However, it is unknown whether (1) ROS production is increased at inotropic stress; and (2) changes in ROS generation influence myocardial contractility physiologically. Herein, we used distinct inotropic stimuli – endothelin-1 (ET-1) and b₁-adrenergic receptor agonist dobutamine – to characterize the possible relationship between endogenous ROS production and regulation of cardiac contractility in intact rat hearts.

Methods: 

Contractility (apicobasal displacement) was measured in Langendorff-perfused isolated rat hearts (male 7-week-old Sprague-Dawley rats, n=182). ROS generation was measured by dihydroethidium-fluorescent dye using confocal microscopy. Phosphorylation status of phospholamban and extracellular signal-regulated kinase (ERK) were determined by Western blot analysis.

Results: 

An increase in ROS production (P<0.05) was observed after ET-1 (1 nmol/L) stimulus, and ET-1 increased developed tension by 45±6%. The rise in ROS production was abolished by ROS scavenger N-acetylcysteine (NAC, 500 micromol/L) and nicotinamide-dinucleotide phosphate (NAD(P)H) oxidase inhibitor apocynin (100 mmol/L) (P<0.05). Moreover, NAC and apocynin attenuated the ET-1-induced inotropic effect by 33% (P<0.001) and 36% (P<0.001), respectively. ET-1 increased ERK phosphorylation (P<0.001). This effect was attenuated by NAC (P<0.01) and apocynin (P<0.05). In contrast, the positive inotropic effect of dobutamine (250 nmol/L) was augmented by NAC and apocynin (P<0.01 and P<0.05). While dobutamine-induced phospholamban phosphorylation (Ser-16; Thr-17) was enhanced by apocynin (P<0.05), dobutamine had no effect on ERK phosphorylation with or without the NAD(P)H oxidase inhibition.

Conclusion: 

NAD(P)H oxidase-derived ROS acts as a regulator of cardiac contractility acutely in intact rat hearts. However, ROS modulate the inotropic responses differentially to characteristically distinct stimuli. Results with ET-1 stimulus reveal that the increase in cardiac contractility via ERK is partly ROS dependent. On the contrary, endogenous ROS generation attenuates the b-adrenergic receptor-induced positive inotropic effect and limits phospholamban phosphorylation.