-Adrenergic Receptor–Stimulated Hypertrophy in Adult Rat Ventricular Myocytes Is Mediated via Thioredoxin-1–Sensitive Oxidative Modification of Thiols on Ras Gabriela M. Kuster, MD; David R. Pimentel, MD; Takeshi Adachi, MD, PhD; Yasuo Ido, MD, PhD; Daniel A. Brenner, MA; Richard A. Cohen, MD; Ronglih Liao, PhD; Deborah A. Siwik, PhD; Wilson S. Colucci, MD Background—-Adrenergic receptor (AR)–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 AR-stimulated hypertrophic signaling is mediated via oxidative modification of Ras thiols. Methods and Results—The effect of AR stimulation on the number of free thiols on Ras was measured with biotinylated iodoacetamide labeling. AR 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, AR-stimulated Ras activation was abolished by TRX1 overexpression and potentiated by azelaic acid. TRX1 overexpression inhibited the AR-stimulated phosphorylation of MEK1/2, ERK1/2, and p90RSK and prevented cellular hypertrophy, sarcomere reorganization, and protein synthesis (versus -galactosidase). Azelaic acid potentiated AR-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 20-fold, TRX1 had no effect. Likewise, the AR-stimulated increase in dichlorofluorescein diacetate fluorescence was abolished with catalase but retained with TRX1. Conclusions—AR-stimulated hypertrophic signaling in adult rat ventricular myocytes is mediated via a TRX1-sensitive posttranslational oxidative modification of thiols on Ras. (Circulation. 2005;111:1192-1198.) Key Words: hypertrophy  reactive oxygen species  sulfhydryl compounds  receptors, adrenergic, alpha  thioredoxin I n adult rat ventricular myocytes (ARVMs) in culture, -adrenergic receptor (AR)–stimulated hypertrophy is mediated via activation of the Ras-Raf-MEK1/2-ERK1/2 signaling pathway. 1,2 We and others have shown that AR- mediated hypertrophic signaling and hypertrophy are medi- ated by reactive oxygen species (ROS). 3–5 Nevertheless, little is known about the mechanism by which ROS initiate hypertrophic signaling. Ras activation is both necessary and sufficient for AR-stimulated protein synthesis in ARVMs. 2 Ras has 4 cysteines with reactive thiol groups that can regulate its activity in response to oxidative modification. 6,7 These observations suggest that ROS could mediate AR- stimulated hypertrophic signaling in ARVMs via the oxida- tive modification of thiols on Ras. Thioredoxin-1 (TRX1) is a cytosolic dithiol-disulfide oxi- doreductase that plays an important role in maintaining intracel- lular thiols in a reduced state. 8 TRX1 has been implicated in the regulation of cell growth in several cell types, including cardiac myocytes. In transgenic mice, myocyte-specific overexpression of TRX1 inhibits myocardial hypertrophy in response to pres- sure overload, whereas overexpression of a dominant negative mutant augments the hypertrophic response. 9 Furthermore, in COS-7 cells, it was shown that TRX1 can regulate the basal level of Ras thiolation. 9 Received June 28, 2004; revision received October 13, 2004; accepted November 12, 2004. From the Cardiovascular Medicine Section, Department of Medicine, and the Myocardial and Vascular Biology Units, Boston University Medical Center, Boston, Mass. Guest Editor for this article was Roberto Bolli, MD. Correspondence to Wilson S. Colucci, MD, Cardiovascular Section, Boston University Medical Center, 88 E Newton St, Boston, MA 02118. E-mail Wilson.colucci@bmc.org © 2005 American Heart Association, Inc. Circulation is available at http://www.circulationaha.org DOI: 10.1161/01.CIR.0000157148.59308.F5 1192 by guest on November 23, 2015 http://circ.ahajournals.org/ Downloaded from