Abstract
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 alpha-actin, atrial natriuretic factor, and the alpha3-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 alpha-actin induction was dependent on Ras but not on p42/44 MAPKs, (b) alpha3 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.
Highlights
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
Since our previous studies suggested that p42/44 mitogen-activated protein kinases (MAPKs) may participate in ouabain-induced down-regulation of the ␣3 subunit of Na/KATPase in cardiac myocytes [7], we wished to determine if these kinases are activated when myocytes are exposed to ouabain concentrations that cause partial inhibition of Naϩ/ Kϩ-ATPase
Myocytes were exposed to 100 M ouabain for various times, and the levels of phosphorylated and activated p42/44 MAPKs were measured by Western blot analysis
Summary
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 growthrelated genes of these myocytes, i.e. those for c-Fos, skeletal ␣-actin, atrial natriuretic factor, and the ␣3-subunit of Na؉/K؉-ATPase. Using cultured neonatal rat cardiac myocytes, we showed [5,6,7] that the same nontoxic concentrations of ouabain that cause partial inhibition of sarcolemmal Naϩ/Kϩ-ATPase and increased intracellular Ca2ϩ, stimulate the hypertrophic growth of these myocytes, and lead to transcriptional regulation of several genes that have been implicated as markers of cardiac hypertrophy. Our findings indicate that while the four ouabain-initiated pathways share an early segment involving Ca2ϩ, calmodulin, and PKC, they subsequently branch out into multiple Ras-dependent and Ras-independent segments only some of which are dependent on p42/44
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