Inhibition of Na/K-ATPase promotes myocardial tumor necrosis factor-alpha protein expression and cardiac dysfunction via calcium/mTOR signaling in endotoxemia

Abstract

Tumor necrosis factor-α (TNF-α) is a major pro-inflammatory cytokine that causes cardiac dysfunction during sepsis. Na/K-ATPase regulates intracellular Ca(2+), which activates mammalian target of rapamycin (mTOR), a regulator of protein synthesis. The aim of this study was to investigate the role of Na/K-ATPase/mTOR signaling in myocardial TNF-α expression during endotoxemia. Results showed that treatment with LPS decreased Na/K-ATPase activity in the myocardium in vivo and in cultured neonatal cardiomyocytes. Inhibition of Na/K-ATPase by ouabain enhanced LPS-induced myocardial TNF-α protein production, but had no effect on TNF-α mRNA expression. More importantly, ouabain further decreased in vivo cardiac function in endotoxemic mice, which was blocked by etanercept, a TNF-α antagonist. LPS-induced reduction in Na/K-ATPase activity was prevented by inhibition of PI3K, Rac1 and NADPH oxidase using LY294002, a dominant-negative Rac1 adenovirus (Ad-Rac1N17) and apocynin, respectively. To assess the role of Rac1 in Ca(2+) handling, Ca(2+) transients in adult cardiomyocytes from cardiomyocyte-specific Rac1 knockout (Rac1(CKO)) and wild-type (WT) mice were determined. LPS increased intracellular Ca(2+) in WT but not in Rac1(CKO) cardiomyocytes. Furthermore, LPS rapidly increased mTOR phosphorylation in cardiomyocytes, which was blocked by Rac1N17 and an inhibitor of calmodulin-dependent protein kinases (CaMKs) KN93, but enhanced by ouabain. Rapamycin, an inhibitor of mTOR suppressed TNF-α protein levels without any significant effect on its mRNA expression or global protein synthesis. In conclusion, myocardial Na/K-ATPase activity is inhibited during endotoxemia via PI3K/Rac1/NADPH oxidase activation. Inhibition of Na/K-ATPase activates Ca(2+)/CaMK/mTOR signaling, which promotes myocardial TNF-α protein production and cardiac dysfunction during endotoxemia.