Peter Kometiani

Intracellular Reactive Oxygen Species Mediate the Linkage of Na+/K+-ATPase to Hypertrophy and Its Marker Genes in Cardiac Myocytes

We showed before that in cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophy and transcriptional regulations of growth-related marker genes through multiple Ca2+-dependent 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 intracellular reactive oxygen species (ROS) in these ouabain-initiated pathways. Ouabain caused a rapid generation of ROS within the myocytes that was prevented by preexposure of cells to N-acetylcysteine (NAC) or vitamin E. These antioxidants also blocked or attenuated the following actions of ouabain: inductions of the genes of skeletal α-actin and atrial natriuretic factor, repression of the gene of the α3-subunit of Na+/K+-ATPase, activation of mitogen-activated protein kinases, activation of Ras-dependent protein synthesis, and activation of transcription factor NF-κB. Induction of c-fos and activation of AP-1 by ouabain were not sensitive to NAC. Ouabain-induced inhibition of active Rb+ uptake through Na+/K+-ATPase and the resulting rise in intracellular Ca2+ were also not prevented by NAC. 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 by NAC. We conclude the following: (a) ROS are essential second messengers within 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 ouabain-induced hypertrophy; (c) increased ROS generation is not a common response of the myocyte to all hypertrophic stimuli; and (d) it may be possible to dissociate the positive inotropic effect of ouabain from its growth-related effects by alteration of the redox state of the cardiac myocyte.

Multiple Signal Transduction Pathways Link Na+/K+-ATPase to Growth-related Genes in Cardiac Myocytes THE ROLES OF Ras AND MITOGEN-ACTIVATED PROTEIN KINASES

We showed before that in neonatal rat cardiac myocytes partial inhibition of Na+/K+-ATPase by nontoxic concentrations of ouabain causes hypertrophic growth and transcriptional regulations of genes that are markers of cardiac hypertrophy. In view of the suggested roles of Ras and p42/44 mitogen-activated protein kinases (MAPKs) as key mediators of cardiac hypertrophy, the aim of this work was to explore their roles in ouabain-initiated signal pathways regulating four growth-related genes of these myocytes,i.e. those for c-Fos, skeletal α-actin, atrial natriuretic factor, and the α3-subunit of Na+/K+-ATPase. Ouabain caused rapid activations of Ras and p42/44 MAPKs; the latter was sustained longer than 90 min. Using high efficiency adenoviral-mediated expression of a dominant-negative Ras mutant, and a specific inhibitor of MAPK kinase (MEK), activation of Ras-Raf-MEK-p42/44 MAPK cascade by ouabain was shown. The effects of the mutant Ras, an inhibitor of Ras farnesylation, and the MEK inhibitor on ouabain-induced changes in mRNAs of the four genes indicated that (a) skeletal α-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) α3 repression was dependent on the Ras-p42/44 MAPK cascade, and (c) induction of c-fos or atrial natriuretic factor gene occurred partly through the Ras-p42/44 MAPK cascade, and partly through pathways independent of Ras and p42/44 MAPKs. All ouabain effects required extracellular Ca2+, and were attenuated by a Ca2+/calmodulin antagonist or a protein kinase C inhibitor. The findings show that (a) signal pathways linked to sarcolemmal Na+/K+-ATPase share early segments involving Ca2+ and protein kinase C, but diverge into multiple branches only some of which involve Ras, or p42/44 MAPKs, or both; and (b) there are significant differences between this network and the related gene regulatory pathways activated by other hypertrophic stimuli, including those whose responses involve increases in intracellular free Ca2+ through different mechanisms.

Positive inotropic effect of ouabain on isolated heart is accompanied by activation of signal pathways that link Na+/K+-ATPase to ERK1/2.

Exposure of cultured rat cardiac myocytes to ouabain is known to cause the interaction of Na+/K+-ATPase with adjacent proteins, leading to activation of multiple signal transduction pathways, regulation of growth-related genes, and hypertrophy. The aim of this work was to determine if the proximal signaling events identified in cultured myocytes also occur in isolated intact hearts of rat and guinea pig in response to positive inotropic doses of ouabain. Langendorff rat heart preparations were exposed to 50 &mgr;M ouabain to produce positive inotropy without toxicity, and assayed for Src kinase, protein kinase C, and extracellular signal-regulated kinases 1 and 2 (ERK1/2). These kinases were rapidly activated by ouabain as in cultured cells. In isolated guinea pig hearts, 1 &mgr;M ouabain caused ERK1/2 activation comparable to the effect of 50 &mgr;M ouabain in rat heart and consistent with the higher ouabain sensitivity of the contractility of guinea pig heart. These data show that the proximal ouabain-induced signal pathways previously noted in cultured cells are not artifacts of dispersion/culturing of myocytes, and are not the peculiar properties of the rat heart with its relatively low ouabain sensitivity. They also suggest that treatment with positive inotropic doses of cardiac glycosides is likely to be associated with changes in the cardiac phenotype.

Regulation of Na/K-ATPase β1-subunit gene expression by ouabain and other hypertrophic stimuli in neonatal rat cardiac myocytes

Partial inhibition of Na/K-ATPase by ouabain causes hypertrophic growth and regulates several early and late response genes, including that of Na/K-ATPase α3 subunit, in cultured neonatal rat cardiac myocytes. The aim of this work was to determine whether ouabain and other hypertrophic stimuli affect Na/K-ATPase β1 subunit gene expression. When myocytes were exposed to non-toxic concentrations of ouabain, ouabain increased β1 subunit mRNA in a dose- and time-dependent manner. Like the α3 gene, β1 mRNA was also regulated by several other well-known hypertrophic stimuli including phenylephrine, a phorbol ester, endothelin-1, and insulin-like growth factor, suggesting involvement of growth signals in regulation of β1 expression. Ouabain failed to increase β1 subunit mRNA in the presence of actinomycin D. Using a luciferase reporter gene that is directed by the 5′-flanking region of the β1 subunit gene, transient transfection assay showed that ouabain augmented the expression of luciferase. These data support the proposition that ouabain regulates the β1 subunit through a transcriptional mechanism. The effect of ouabain on β1 subunit induction, like that on α3 repression, was dependent on extracellular Ca2+ and on calmodulin. Inhibitions of PKC, Ras, and MEK, however, had different quantitive effects on ouabain-induced regulations of β1 and α3 subunits. The findings show that partial inhibition of Na/K-ATPase activates multiple signaling pathways that regulate growth-related genes, including those of two subunit isoforms of Na/K-ATPase, in a gene-specific manner.