Increased Activity of NADPH Oxidase Contributes to Enhanced LV Myocyte Contraction in nNOS−/− Mice

Abstract

Superoxide production from NADPH oxidases has increasingly been shown to play an important role in myocardial signalling. The activity of myocardial NADPH oxidases is known to be increased in the failing myocardium; however, whether this is a compensatory or maladaptive mechanism remains to be established. Gene deletion of the neuronal nitric oxide synthase (nNOS) is associated with an increase in myocardial superoxide production and with enhanced inotropy. Here we tested whether nNOS gene deletion leads to an increase in myocardial NADPH oxidase activity, which - in turn - causes a superoxide (O2-)-dependent increase in contraction.As expected, O2- production (measured by lucigenin 5μmol/L -enhanced chemiluminescence) was greater in nNOS−/− LV myocytes than in their wild type littermates (nNOS+/+). Pre-incubation of LV myocytes with the NADPH oxidase inhibitor apocynin (100 μmol/L, 30 min) reduced the level of O2- in nNOS−/− myocytes only, thereby abolishing the difference between genotypes. In agreement with these findings, apocynin significantly reduced cell shortening (%, field stimulation at 3Hz, 35°C) only in nNOS−/− myocytes. Inhibition of protein kinase A (amide 14-22, PKI, 2 μmol/L) reduced contraction to a larger extent in nNOS−/−. The effects of PKA inhibition were abolished after pre-incubation with apocynin. NADPH oxidase stimulation by endothelin-1 (ET-1, 10 nM, 5-10 min) caused an increase in cell shortening in both nNOS−/− and nNOS+/+ myocytes, which was abolished by apocynin. PKI significantly reduced the effect of ET-1 in both genotypes.Taken together, these findings suggest that nNOS-derived NO may tonically inhibit the activity of NADPH oxidase in murine LV myocytes and indicate that production of O2- by this oxidase system may account for the PKA-dependent increase in cell shortening in nNOS−/− mice.