Alpha-adrenergic receptor-stimulated hypertrophy in adult rat ventricular myocytes is mediated via thioredoxin-1-sensitive oxidative modification of thiols on Ras

Abstract

Background: Alpha-adrenergic receptor (alphaAR)-stimulated hypertrophy in adult rat ventricular myocytes is mediated by reactive oxygen species-dependent activation of the Ras-Raf-MEK1/2-ERK1/2 signaling pathway. Because Ras is known to have redox-sensitive cysteine residues, we tested the hypothesis that alphaAR-stimulated hypertrophic signaling is mediated via oxidative modification of Ras thiols.

Methods and results: The effect of alphaAR stimulation on the number of free thiols on Ras was measured with biotinylated iodoacetamide labeling. alphaAR stimulation caused a 48% decrease in biotinylated iodoacetamide-labeled Ras that was reversed by dithiothreitol (10 mmol/L), indicating a decrease in the availability of free thiols on Ras as a result of an oxidative posttranslational modification. This effect was abolished by adenoviral overexpression of thioredoxin-1 (TRX1) and potentiated by the TRX reductase inhibitor azelaic acid. Likewise, alphaAR-stimulated Ras activation was abolished by TRX1 overexpression and potentiated by azelaic acid. TRX1 overexpression inhibited the alphaAR-stimulated phosphorylation of MEK1/2, ERK1/2, and p90RSK and prevented cellular hypertrophy, sarcomere reorganization, and protein synthesis (versus beta-galactosidase). Azelaic acid potentiated alphaAR-stimulated protein synthesis. Although TRX1 can directly reduce thiols, it also can scavenge ROS by increasing peroxidase activity. To examine this possibility, peroxidase activity was increased by transfection with catalase, and intracellular reactive oxygen species were measured with dichlorofluorescein diacetate fluorescence. Although catalase increased peroxidase activity approximately 20-fold, TRX1 had no effect. Likewise, the alphaAR-stimulated increase in dichlorofluorescein diacetate fluorescence was abolished with catalase but retained with TRX1.

Conclusions: AlphaAR-stimulated hypertrophic signaling in adult rat ventricular myocytes is mediated via a TRX1-sensitive posttranslational oxidative modification of thiols on Ras.