Intracellular reactive oxygen species mediate the linkage of Na+/K+-ATPaseto hypertrophy and its marker genes in cardiac myocytes

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPa se by nontoxic concentrations of ouabain causes hypertrophy and transcripti onal regulations of growth-related marker genes through multiple Ca2+-depen dent signal pathways many of which involve Ras and p42/44 mitogen-activated protein kinases, The aim of this work was to explore the roles of intracel lular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ou abain caused a rapid generation of ROS within the myocytes that was prevent ed by preexposure of cells to N-acetyl-cysteine (NAC) or vitamin E, These a ntioxidants also blocked or attenuated the following actions of ouabain: in ductions of the genes of skeletal alpha-actin and atrial natriuretic factor , repression of the gene of the alpha(3)-subunit of Na+/K+-ATPase, activati on of mitogen-activated protein kinases, activation of Ras-dependent protei n synthesis, and activation of transcription factor NF-kappa B. Induction o f c-fos and activation of AP-I by ouabain were not sensitive to NAG. Ouabai n-induced inhibition of active Rb+ uptake through Na+/K+-ATPase and the res ulting rise in intracellular Ca2+ were also not prevented by NAG. A phorbol ester that also causes myocyte hypertrophy did not increase ROS generation , and its effects on marker genes and protein synthesis were not affected b y NAG. We conclude the following: (a) ROS are essential second messengers w ithin some but not all signal pathways that are activated by the effect of ouabain on Na+/K+-ATPase; (b) the ROS-dependent pathways are involved in ou abain-induced hypertrophy; (c) increased ROS generation is not a common res ponse of the myocyte to all hypertrophic stimuli; and (d) it may be possibl e to dissociate the positive inotropic effect of ouabain from its growth-re lated effects by alteration of the redox state of the cardiac myocyte.