Published online before print February 21, 2005, doi: 10.1161/01.CIR.0000157148.59308.F5
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(Circulation. 2005;111:1192-1198.)
© 2005 American Heart Association, Inc.
Molecular Cardiology |
-Adrenergic Receptor–Stimulated Hypertrophy in Adult Rat Ventricular Myocytes Is Mediated via Thioredoxin-1–Sensitive Oxidative Modification of Thiols on Ras
From the Cardiovascular Medicine Section, Department of Medicine, and the Myocardial and Vascular Biology Units, Boston University Medical Center, Boston, Mass.
Correspondence to Wilson S. Colucci, MD, Cardiovascular Section, Boston University Medical Center, 88 E Newton St, Boston, MA 02118. E-mail Wilson.colucci{at}bmc.org
Received June 28, 2004; revision received October 13, 2004; accepted November 12, 2004.
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Abstract |
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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 ß-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.
Key Words: hypertrophy • reactive oxygen species • sulfhydryl compounds • receptors, adrenergic, alpha • thioredoxin
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Introduction |
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In 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 mediated 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 oxidative modification of thiols on Ras. Thioredoxin-1 (TRX1) is a cytosolic dithiol-disulfide oxidoreductase that plays an important role in maintaining intracellular 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 pressure 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
It is not known whether a hypertrophic stimulus can cause oxidative posttranslational modification of Ras thiols in cardiac myocytes. Therefore, the goal of the present study was to test the hypothesis that AR-stimulated hypertrophy is mediated via the oxidative modification of thiols on Ras. The abundance of free thiols on Ras was measured by labeling with biotinylated iodoacetamide (BIAM). AR stimulation decreased the availability of free thiols on Ras; this effect was reversed by the reducing agent dithiothreitol (DTT), thus indicating that AR stimulation caused an oxidative posttranslational modification. The AR-stimulated decrease in free thiols was abolished by adenoviral overexpression of TRX1 and potentiated by the TRX reductase inhibitor azelaic acid, indicating that this effect was TRX1 sensitive. Finally, the functional importance of the TRX1-sensitive Ras thiol modification was assessed by measuring the effects of TRX1 and azelaic acid on AR-stimulated myocyte hypertrophy and hypertrophic signaling.
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Methods |
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Cell Culture
ARVMs were isolated from the hearts of adult (200 to 220 g) male Sprague-Dawley rats as described previously.10 Cells were plated at a nonconfluent density of 50 to 75 cells/mm2 on plastic culture dishes or glass coverslips precoated with laminin (1 µg/cm2) and kept at 37°C in ACCT medium (DMEM, BSA 2 mg/mL, L-carnitine 2 mmol/L, creatinine 5 mmol/L, taurine 5 mmol/L, penicillin 100 IU/mL, streptomycin 10 µg/mL) for 2 hours before adenoviral infection was performed.
Cell Treatments
L-Norepinephrine (1 µmol/L; Sigma) was added for 5 minutes (free Ras thiols, signaling), 48 hours (leucine incorporation, immunocytochemistry), or 30 minutes (intracellular ROS) before measurements. DL-Propranolol (2 µmol/L; Sigma) was added 30 minutes before L-norepinephrine. In some experiments, the TRX reductase inhibitor azelaic acid (10 µmol/L; Sigma)11 was added 16 hours before AR stimulation. All plates were supplemented with ascorbic acid (100 µmol/L; Sigma) to prevent oxidation of L-norepinephrine.
Adenoviral Constructs
An adenoviral plasmid expressing human TRX1 (ATCC, accession No. BC003377) was created by use of Gateway technology (Invitrogen). The TRX1 gene was transferred via BP and LR reactions to the adenoviral vector (Clontech) in which a Gateway Cassette (Invitrogen) had been inserted downstream of the cytomegalovirus promoter site. The ß-galactosidase (ß-gal) and catalase (CAT) vectors were constructed as described by He et al.12 The adenoviral plaques were amplified in HEK 293 cells and purified with a double cesium chloride gradient. The viral titer was determined by the TCID50 method. Thirty-six hours before drug treatments, ARVMs were infected with the adenoviruses at a multiplicity of infection of 50/100.
Ras Free Thiols
Free reactive thiols were measured with BIAM (Molecular Probes) by a modification of the technique of Kim et al.13 Briefly, cells were lysed in buffer (1% NP 40, 0.25% DOC, 50 mmol/L PIPES, 100 µmol/L DTPA, 150 mmol/L NaCl, pH 6.5) containing 100 µmol/L BIAM. The lysates were separated by centrifugation at 14 000 rpm; ß-mercaptoethanol (50 mmol/L) was added to stop further thiol labeling; and the proteins were passed through a PD-10 Sephadex-G25 column to eliminate excess free BIAM. BIAM-labeled proteins were gathered with streptavidin-sepharose beads (50 µL) overnight, washed 4 times with lysis buffer, and separated from the beads by adding Laemmli buffer containing 5 mol/L urea. BIAM-labeled Ras was detected by Western blotting with a monoclonal anti-Ras antibody. In some experiments, DTT (10 mmol/L) was added to the lysis buffer before processing as described above.
Ras Activation Assay
Activated Ras was detected by a Ras activation assay kit (Upstate Biotechnology) according to the manufacturer’s instructions as previously described.4 Briefly, cell lysates (500 µg) were incubated at 4°C overnight with an agarose conjugate Ras-GTP affinity probe corresponding to the human Ras binding domain of Raf-1. The precipitates were resolved on a 4% to 20% SDS-PAGE and detected by Western blotting with an anti-Ras antibody.
Western Blotting
MEK1/2, ERK1/2, and p90RSK phosphorylation were assessed as previously described1 with anti–phospho-MEK1/2, anti-phospho-p44/42 MAP kinase, and anti–phospho-p90RSK antibodies (all from Cell Signaling). TRX1 was assessed with a polyclonal anti-human TRX1 antibody (BD Biosciences).
Immunocytochemistry and Cell Size
Cells were washed with PBS, fixed with 3.7% buffered formaldehyde for 30 minutes, and permeabilized with methanol at –20°C for 20 minutes. Cells were then treated with 5% BSA for 1 hour at room temperature and incubated with a monoclonal tetramethylrhodamine-5- (and 6)-isothiocyanate (TRITC)–conjugated (Pierce) anti–-sarcomeric actinin antibody for -sarcomeric actinin (clone EA-53, Sigma) at 37°C for another hour. Myocyte surface area was assessed with semiautomatic computer-assisted planimetry (Bioquant) from 2D images of unstained cells.
Leucine Incorporation
The cells were plated on 6-well laminin-coated dishes, and protein synthesis was measured as described previously.14
Peroxidase Activity
Total cellular peroxidase activity was measured by spectrophotometry with 14 mmol/L H2O2 in a 15-mmol/L potassium phosphate buffer solution (pH 7) as described.15
Intracellular ROS
Intracellular ROS were assessed with the ROS-sensitive fluorophore dichlorofluorescein diacetate (DCF) (Molecular Probes) as described previously.16 Briefly, cells were incubated with 20 µmol/L DCF for 30 minutes, and fluorescence was visualized and quantified with epifluorescent microscopy and video imaging (Bioquant, version 2.5).
Statistical Analysis
All data are presented as mean±SEM. Differences across multiple conditions were tested by 1-way ANOVA. Comparisons between conditions were tested by Student unpaired t test with Bonferroni correction for multiple comparisons. A value of P<0.05 was considered significant.
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Results |
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AR Stimulation Decreases Free Ras ThiolsTo test the hypothesis that
AR-stimulated hypertrophic signaling involves the modification of thiols on Ras, free thiols on Ras were measured by labeling with BIAM.13 AR stimulation (5 minutes) caused a 48% decrease in biotinylated Ras, indicating a decrease in the availability of free thiols resulting from a posttranslational modification. The AR-stimulated decrease in BIAM labeling was reversed by the addition of DTT to the lysis buffer, indicating that it is due to an oxidative modification (Figure 1A).
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P<0.01, 
P<0.001).
The AR-Stimulated Decrease in Free Ras Thiols Is TRX1 Sensitive
TRX1 was overexpressed by infection with an adenoviral vector for 36 hours. Under these conditions, TRX1 protein expression increased 6-fold, from 8±1 to 44±2 arbitrary units (P<0.0001 versus ß-gal). AR stimulation alone for up to 48 hours had no effect on TRX1 expression (data not shown). TRX1 overexpression abolished the AR-stimulated decrease in free Ras thiols (Figure 1B), suggesting that the decrease in free thiols is due to an oxidative modification that is sensitive to TRX1. To examine the role of endogenous TRX, TRX reductase was inhibited with azelaic acid.11 Azelaic acid decreased the abundance of both basal and AR-stimulated free Ras thiols (Figure 1C).
Overexpression of TRX1 Inhibits AR-Stimulated Ras Activation
AR stimulation (5 minutes) increased Ras activity by 2-fold (Figure 2). TRX1 overexpression had no effect on basal Ras activity but abolished the AR-stimulated increase (Figure 2A). Conversely, inhibition of TRX with azelaic acid tended to increase basal Ras activity and significantly potentiated the AR-stimulated increase (Figure 2B).
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Overexpression of TRX1 Inhibits AR-Stimulated Downstream Hypertrophic Signaling
TRX1 overexpression abolished AR-stimulated phosphorylation of MEK1/2 and inhibited AR-stimulated phosphorylation of both ERK1/2 and its substrate, p90RSK (Figure 3).
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Overexpression of TRX1 Inhibits AR-Stimulated Hypertrophy in ARVMs
We have previously shown that AR stimulation (48 hours) causes cellular hypertrophy that is associated with sarcomere reorganization and increased protein synthesis.3 TRX1 overexpression abolished both AR-stimulated sarcomere reorganization and hypertrophy (Figure 4A and 4B). Expression of ß-gal had no effect on basal or AR-stimulated hypertrophy or sarcomere reorganization compared with uninfected control cells (data not shown).
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Leucine incorporation was measured to assess protein synthesis. In ß-gal–expressing cells, AR stimulation increased leucine incorporation by 44±2%. TRX1 overexpression decreased basal leucine incorporation by 63±3% and abolished the response to AR stimulation (–95±9% versus ß-gal) (Figure 4C). The decrease in basal leucine incorporation was not due to cell loss because TRX1 overexpression had no effect on cell number (TRX1, 54±7 cells/mm2; ß-gal, 55±3 cells/mm2; P=NS; n=3). Likewise, TRX1 overexpression had no effect on total cellular protein, which was 104±3% of that in ß-gal–expressing cells (P=NS; n=4). In contrast, inhibition of TRX with azelaic acid caused a 49±3% augmentation of AR-stimulated leucine incorporation (P<0.05, n=3). TRX1 overexpression had no effect on leucine incorporation in response to either 3% or 10% serum (data not shown), indicating that inhibition of AR-stimulated protein synthesis is not due to a generalized effect on protein synthesis.
Effect of TRX1 Overexpression on Peroxidase Activity and Intracellular ROS
TRX1 might inhibit oxidative thiol modification directly via an interaction with the thiol group or indirectly by increasing peroxidase activity. TRX1 overexpression had no effect on peroxidase activity in ARVMs, whereas adenoviral overexpression of CAT increased peroxidase activity 20-fold (Figure 5). AR stimulation increased intracellular ROS, as assessed by DCF, by 117±15% (Figure 6A and 6B). Although both TRX1 and CAT overexpression decreased basal DCF, the AR-stimulated increase was prevented by CAT but not TRX1 overexpression.
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P<0.01 vs ß-gal). |
Discussion |
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This study provides several new observations about the redox regulation of hypertrophic signaling in adult cardiomyocytes. First,
AR stimulation decreased the availability of free thiols on Ras, and this effect was reversed by the reducing agent DTT. Second, the AR-stimulated decrease in Ras free thiols was prevented by overexpression of TRX1 and potentiated by inhibition of TRX with azelaic acid. Third, in parallel with the changes in free thiols, the AR-stimulated activation of Ras was prevented by overexpression of TRX1 and potentiated by azelaic acid. Finally, TRX1 inhibited AR-stimulated activation of the Ras-associated hypertrophic signaling pathway and myocyte hypertrophy, whereas azelaic acid potentiated AR-stimulated hypertrophy.
We previously demonstrated in ARVMs that AR-stimulated hypertrophy is associated with ROS-mediated activation of Ras.1,4 Recently, Wang and Proud2 showed that Ras activation is both necessary and adequate for AR-stimulated protein synthesis in ARVMs. Ras has 4 cysteines with reactive thiol groups that can regulate its activity in response to oxidative modifications such as S-nitrosylation or S-glutathiolation.6,7 Free (ie, reduced) thiols react with iodoacetamide; therefore, the availability of free thiols can be quantified by use of BIAM to label and separate reduced thiols.13 AR stimulation caused a rapid (5 minutes) 50% decrease in the abundance of free Ras thiols, indicating a posttranslational modification. The AR-stimulated decrease was reversed by DTT, indicating that the modification was oxidative.
To clarify the functional consequences of the AR-stimulated Ras modification, TRX1 was overexpressed. TRX1 is an oxidoreductase that plays an important role in maintaining intracellular thiols in a reduced state.8 TRX1 overexpression prevented the AR-stimulated decrease in free Ras thiols and the activation of Ras, indicating that both effects are mediated via a TRX1-sensitive mechanism. Likewise, TRX1 overexpression inhibited AR-stimulated activation of the MEK/ERK signaling cascade that plays a major role in mediating hypertrophy in adult rat cardiac myocytes.1 Although TRX1 overexpression abolished AR-stimulated Ras activation, inhibition of ERK was not complete. This observation is consistent with prior observations suggesting that there is a small amount of Ras-independent AR-stimulated ERK1/2 activation in adult rat cardiac myocytes.2 Conversely, inhibition of endogenous TRX with azelaic acid potentiated both the AR-stimulated decrease in free thiols and the AR-stimulated activation of Ras.
These effects of TRX overexpression and inhibition, together with the effect of DTT, support our hypothesis that AR-stimulated activation of Ras requires an oxidative modification of thiols. This thesis is also consistent with our recent demonstration that the oxidative modification of Cyc118 of Ras via S-glutathiolation mediates angiotensin-induced hypertrophic signaling in vascular smooth muscle cells17 and the demonstration by Yamamoto et al9 that overexpression of dominant negative TRX1 increases the basal level of Ras thiolation in COS-7 cells.
TRX can regulate the intracellular redox state by 2 mechanisms. First, TRX can directly catalyze the reduction of protein thiol groups.18–20 Second, the TRX system, consisting of TRX, TRX reductase, and NADPH, can scavenge ROS by supplying electrons for TRX peroxidase and other peroxidases, leading to increased peroxidase activity.21,22 Our data show that TRX1 overexpression had no measurable effect on cellular peroxidase activity. Likewise, although TRX1 decreased basal and AR-stimulated intracellular ROS levels, it had no effect on the AR-stimulated increase. The decrease in ROS with TRX1 likely reflects a direct chemical interaction between H2O2 and reduced sulfhydryl groups on TRX1 rather than increased peroxidase activity. Taken together, these observations suggest that TRX1 exerts its antihypertrophic effect by decreasing the oxidative posttranslational modification of Ras thiol groups. The data are consistent with the thesis that TRX1 inhibits AR-stimulated hypertrophy by protecting Ras thiols from oxidative modification. Although we cannot exclude the possibility that TRX1 also inhibits AR-stimulated signaling proximal to Ras, it seems unlikely because TRX1 did not inhibit the AR-stimulated increase in ROS as measured by DCF and because we have found no effect of TRX1 overexpression on AR-stimulated NADPH oxidase activity (data not shown).
TRX1 overexpression decreased AR-stimulated myocyte hypertrophy, as evidenced by inhibition of the AR-stimulated increase in myocyte size, sarcomere reorganization, and protein synthesis. Conversely, inhibition of endogenous TRX with azelaic acid potentiated AR-stimulated protein synthesis. These observations are consistent with those of Yamamoto et al,9 who found that cardiac-specific transgenic overexpression of TRX1 in mice inhibited the hypertrophic effect of aortic banding and conversely that overexpression of a TRX1 dominant negative mutant potentiated the hypertrophic response. We found that TRX1 also decreased basal leucine incorporation in unstimulated myocytes: The decrease in leucine incorporation was not associated with a reduction in cell number, indicating that it was due to a decrease in basal protein synthesis rather than cell loss. The TRX1-induced decrease in basal protein synthesis was not associated with a decrease in cell size. This observation raises the possibility that TRX1 also decreased protein degradation and is consistent with the demonstration that oxidative thiol modifications can target proteins for degradation by the ubiquitin-proteasome pathway.23 In addition, we found that TRX1-overexpressing myocytes had a preserved protein synthetic response to serum, further indicating that the effects of TRX1 on both basal and AR-stimulated protein synthesis are not due to a generalized decrease in cellular synthetic function.
It should be noted that the potent antihypertrophic effects of TRX1 found in our study in adult myocytes and by Yamamoto et al9 in mice differ from the findings of Yoshioka et al,24 who found that in vitro overexpression of TRX1 increased basal and AR-stimulated protein synthesis in neonatal rat cardiac myocytes, whereas overexpression of TRX-interacting protein, an endogenous inhibitor of TRX1, attenuated the hypertrophic response. They also found that in vivo inhibition of TRX1 with TRX-interacting protein decreased the hypertrophic response to pressure overload. Given the multiple actions of and substrates for TRX1, these disparate results may be due to differences related to cell type, cell species, and/or developmental stage. For example, although we have shown that TRX1 can inhibit Ras-mediated hypertrophic signaling, TRX1 can also activate transcription factors (eg, NF-
B, AP-1), interact with signaling proteins (eg, ASK, PKC), and possibly bind to a cell surface receptor.25–32
Taken together, our findings indicate that AR stimulation causes the oxidative posttranslational modification of Ras in adult cardiac myocytes. Overexpression of TRX1 attenuated and inhibition of TRX potentiated the AR-stimulated decrease in free thiols, Ras activation, and myocyte hypertrophy, thus supporting the functional relevance of the posttranslational Ras modification. These observations identify a specific molecular site at which ROS act to mediate myocyte hypertrophy. Thus, modulation of intracellular thiol redox state may play a role in the pathophysiology and/or therapy of myocardial remodeling.
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Acknowledgments |
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This work was supported by NIH National Heart, Lung and Blood Institute sponsoring of the Boston University Cardiovascular Proteomics Center (contract HHSN268200248178C) and grants HL-61639 and HL-20612 (Dr Colucci) and by grants from the American Heart Association (Drs Siwik, Pimentel, and Adachi), Swiss National Science Foundation (Dr Kuster), and Kilo Diabetes Research Foundation (Dr Ido). We thank Xinxin Guo, Kara Clemente, and Jing Wang for their expert technical assistance.
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Footnotes |
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Guest Editor for this article was Roberto Bolli, MD.
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References |
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 A. Gericke, P. Martinka, I. Nazarenko, P. B. Persson, and A. Patzak Impact of {alpha}1-adrenoceptor expression on contractile properties of vascular smooth muscle cells Am J Physiol Regulatory Integrative Comp Physiol, September 1, 2007; 293(3): R1215 - R1221. [Abstract] [Full Text] [PDF]
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 A. Martinez-Ruiz and S. Lamas Signalling by NO-induced protein S-nitrosylation and S-glutathionylation: Convergences and divergences Cardiovasc Res, July 15, 2007; 75(2): 220 - 228. [Abstract] [Full Text] [PDF]
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 E. Takimoto and D. A. Kass Role of Oxidative Stress in Cardiac Hypertrophy and Remodeling Hypertension, February 1, 2007; 49(2): 241 - 248. [Full Text] [PDF]
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J. M. Zimmet and J. M. Hare Nitroso-Redox Interactions in the Cardiovascular System Circulation, October 3, 2006; 114(14): 1531 - 1544. [Full Text] [PDF]
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C. E. Murdoch, M. Zhang, A. C. Cave, and A. M. Shah NADPH oxidase-dependent redox signalling in cardiac hypertrophy, remodelling and failure Cardiovasc Res, July 15, 2006; 71(2): 208 - 215. [Abstract] [Full Text] [PDF]
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 A. Cave, D. Grieve, S. Johar, M. Zhang, and A. M Shah NADPH oxidase-derived reactive oxygen species in cardiac pathophysiology Phil Trans R Soc B, December 29, 2005; 360(1464): 2327 - 2334. [Abstract] [Full Text] [PDF]
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V. Senthil, S. N. Chen, N. Tsybouleva, T. Halder, S. F. Nagueh, J. T. Willerson, R. Roberts, and A.J. Marian Prevention of Cardiac Hypertrophy by Atorvastatin in a Transgenic Rabbit Model of Human Hypertrophic Cardiomyopathy Circ. Res., August 5, 2005; 97(3): 285 - 292. [Abstract] [Full Text] [PDF]
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