Differential regulation of Na/K-ATPase alpha-subunit isoform gene expressions in cardiac myocytes by ouabain and other hypertrophic stimuli.

We showed before that partial inhibition of Na/K-ATPase by non-toxic concentrations of ouabain caused hypertrophic growth of neonatal rat cardiac myocytes, and induced several early- and late-response genes that are markers of cardiac hypertrophy. The aim of this study was to determine if the genes of the alpha-subunit isoforms of Na/K-ATPase were among those regulated by ouabain; and if so, to begin the characterization of the pathways regulating these genes. When neonatal myocytes, expressing alpha1- and alpha3-isoform messages, were exposed to 5-100 micro M ouabain, alpha1 mRNA was not affected, but alpha3 mRNA was decreased in a dose- and time-dependent manner. Ouabain-induced down-regulation of alpha3 mRNA was accompanied by a decrease in alpha3-protein content in these myocytes. There was a significant correlation between ouabain effects on alpha3-repression and skeletal alpha-actin induction; also, ouabain's transcriptional effects on both genes were antagonised by retinoic acid. These findings suggested the association of alpha3 repression with ouabain-induced hypertrophy. Phenylephrine and a phorbol ester, two hypertrophic stimuli that do not inhibit Na/K-ATPase, also down-regulated alpha3 mRNA without affecting alpha1 mRNA, suggesting that alpha3-repression is a common feature of the hypertrophic phenotype in these myocytes. Ouabain-induced repression of alpha3 required the influx of extracellular Ca2+, and was antagonized by inhibitors of protein kinase C, Ca2+-calmodulin kinase, and mitogen-activated protein kinase but not by inhibition of protein kinase A. These data, and prior findings on the mechanisms of hypertrophic effects of phenylephrine and phorbol esters, suggest that transcriptional repression of alpha3 by ouabain and other hypertrophic stimuli involves a common step regulated by a mitogen-activated protein kinase.