AJP - Heart AJP: Regulatory, Integrative and Comparative Physiology
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Heart Circ Physiol 295: H416-H424, 2008. First published May 30, 2008; doi:10.1152/ajpheart.91446.2007
0363-6135/08 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/1/H416    most recent
91446.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X.
Right arrow Articles by Rozanski, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X.
Right arrow Articles by Rozanski, G. J.

Regulation of Kv4 channel expression in failing rat heart by the thioredoxin system

Xun Li,1,2 Kang Tang,1 Bin Xie,1 Shumin Li,1 and George J. Rozanski1,3

1Department of Cellular and Integrative Physiology, University of Nebraska Medical Center, Omaha, Nebraska; 2Department of Cardiology, The First Affiliated Hospital, Suzhou University, Suzhou, Jiangsu, People's Republic of China; and 3Center for Redox Biology, University of Nebraska-Lincoln, Lincoln, Nebraska

Submitted 12 December 2007 ; accepted in final form 22 May 2008


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Redox imbalance elicited by oxidative stress contributes to pathogenic remodeling of ion channels that underlies arrhythmogenesis and contractile dysfunction in the failing heart. This study examined whether the expression of K+ channels in the remodeled ventricle is controlled by the thioredoxin system, a principal oxidoreductase network regulating redox-sensitive proteins. Ventricular dysfunction was induced in rats by coronary artery ligation, and experiments were conducted 6–8 wk postinfarction. Biochemical assays of tissue extracts from infarcted hearts showed that thioredoxin reductase activity was decreased by 32% from sham-operated controls (P < 0.05), whereas thioredoxin activity was 51% higher postinfarction (P < 0.05). These differences in activities paralleled changes in protein abundance as determined by Western blot analysis. However, whereas real-time PCR showed thioredoxin reductase mRNA levels to be significantly decreased postinfarction, thioredoxin mRNA was not altered. In voltage-clamp studies of myocytes from infarcted hearts, the characteristic downregulation of transient-outward K+ current density was reversed by exogenous pyruvate (5 mmol/l), and this effect was blocked by the specific inhibitors of the thioredoxin system: auranofin or 13-cis-retinoic acid. Real-time PCR and Western blot analyses of myocyte suspensions from infarcted hearts showed that pyruvate increased mRNA and protein abundance of Kv4.2 and Kv4.3 channel {alpha}-subunits as well as the accessory protein KChIP2 when compared with time-matched, untreated cells (P < 0.05). The pyruvate-induced increase in Kv4.x expression was blocked by auranofin, but the upregulation of KChIP2 expression was not affected. These data suggest that the expression of Kv4.x channels is redox-regulated by the thioredoxin system, which may be a novel therapeutic target to reverse or limit electrical remodeling of the failing heart.

potassium channels; heart failure; electrical remodeling; transient-outward potassium current

VENTRICULAR DYSFUNCTION induced by different disease states is linked to increased production of reactive oxygen species (ROS) and oxidative damage of myocytes (31). It is proposed that myocardial disease and pressure overload lead to a variety of compensatory processes that are known stimuli for ROS production, such as neurohumoral activation, leukocyte infiltration, increased prostaglandin synthesis, and increased O2 consumption (8, 11). Among the many functional changes elicited in the diseased and failing heart is a pathogenic process of electrical remodeling, which is often characterized by the downregulation of K+ channel activity and which is proposed to contribute to contractile dysfunction and increased incidence of arrhythmic sudden death in heart failure patients (3, 22). Although the mechanisms underlying electrical remodeling are not well understood, it is postulated that a significant decrease in K+ channel activity contributes to arrhythmogenic abnormalities in repolarization (3) and cellular alterations in Ca2+ handling that impact contractile function (35).

Recent data from our laboratory suggest that oxidative stress participates in K+ channel remodeling in the infarcted rat heart through redox-mediated mechanisms (26, 27, 28). Indeed, an important consequence of oxidative stress in the myocardium is a shift in the redox state of cellular proteins, which is characterized by the formation of distinct molecular intermediates of the sulfhydryl group of cysteine residues (23). The unique biochemistry of the sulfhydryl group plays an important role in modulating cell function, since its redox state often determines the structure and activity of essential proteins involved in cell homeostasis. The regulation of the redox state of protein sulfhydryls normally involves the activities of thiol-disulfide oxidoreductases that belong to the thioredoxin superfamily (7, 9, 23, 37). The principal cytosolic oxidoreductase network in mammalian cells is the thioredoxin system, which is composed of thioredoxin-1 (Trx), thioredoxin reductase-1 (TrxR), and NADPH (1, 9, 37). A recently identified thioredoxin-interacting protein (Txnip) also negatively regulates the oxidoreductase activity of Trx by binding to its reduced, active form (34, 38). In parallel with the Trx system, a functionally related but less robust glutaredoxin system also regulates cellular redox status and is composed of glutaredoxin-1 (Grx), GSH, glutathione reductase (GR), and NADPH (7). Both systems function within the cell to maintain proteins in the reduced state and to detoxify ROS. In terms of protein regulation, the Trx system is proposed to catalyze the reduction of inter- and intramolecular disulfides, whereas the Grx system predominantly catalyzes the reduction of protein-mixed disulfides (7, 9, 23, 37). Thus these oxidoreductase systems control the biological function of proteins that are sensitive to oxidative stress conditions.

The purpose of this study was to examine the role of the endogenous Trx system in controlling pathogenic electrical remodeling of the ventricle after myocardial infarction (MI). Our data suggest that the Trx system regulates the expression of Kv4.x channels that characteristically undergo remodeling in disease states associated with oxidative stress. The sensitivity of channel expression to stress-induced alterations in the cell redox state identifies oxidoreductase systems as novel therapeutic targets to limit the impact of electrical remodeling in the failing heart.


   METHODS
TOP
 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Post-MI rat model: isolation of cardiac myocytes. All procedures used for this study were approved by University of Nebraska Medical Center Institutional Animal Care and Use Committee and conducted according to the Guide for the Care and Use of Laboratory Animals, published by the National Institutes of Health (NIH Publication No. 85-23, Revised 1996).

A post-MI model of ventricular dysfunction was used in the present investigation as described previously (26, 28). Briefly, male Sprague-Dawley rats (180–200 g) were intubated and artificially ventilated under Brevital (methohexital sodium) anesthesia at 50 mg/kg ip. A left thoracotomy was performed and the left coronary artery ligated by a suture positioned between the pulmonary artery outflow tract and the left atrium. The thorax was closed, and the rats were allowed to recover for 6–8 wk before experimentation. This ligation protocol produces infarcts of 30–40% of the left ventricular free wall and physiological signs of heart failure after several weeks (26). Sham-operated animals that served as controls underwent the same surgical procedure but were not subjected to coronary artery ligation.

Six to eight weeks after MI or sham operation, rats were given an overdose of pentobarbital sodium (100 mg/kg ip), and the hearts were excised to obtain tissue samples or to isolate ventricular myocytes. For the latter, myocytes were dissociated from Langendorff-perfused hearts by a collagenase digestion procedure described previously (26, 28). Dispersed myocytes from surviving regions of the left ventricle and septum were suspended in DMEM and stored in an incubator at 37°C until used, usually within 6 h of isolation. For the study of ionic currents (see Recording techniques), aliquots of myocytes were transferred to a cell chamber on the stage of an inverted microscope and superfused with a solution containing (in mmol/l) 138 NaCl, 4.0 KCl, 1.2 MgCl2, 1.8 CaCl2, 18 glucose, 5 HEPES, and 0.5 CdCl2 (to block Ca2+ channels) (pH 7.4). Unless stated otherwise, all chemical reagents used in these studies were purchased from Sigma-Aldrich (St. Louis, MO).

Recording techniques. Ionic currents were recorded using the whole cell configuration of the patch-clamp technique. Briefly, borosilicate glass capillaries were pulled (Sutter Instruments, Novato, CA) to an internal tip diameter of 1 to 2 µm and filled with a pipette solution containing (in mmol/l) 135 KCl, 3 MgCl2, 10 HEPES, 3 Na2-ATP, 10 EGTA, and 0.5 Na-GTP (pH 7.2). Filled pipettes with a resistance of 2–4 M{Omega} were coupled to a patch-clamp amplifier (Axopatch 200B, Molecular Devices, Sunnyvale, CA). After the correction of the liquid junction potential and the creation of a G{Omega} seal, the membrane within the pipette was ruptured and at least 5 min were allowed for the contents of the pipette and cytoplasm to equilibrate. A computer program (pClamp, Molecular Devices) controlled command potentials and acquired current signals that were filtered at 2 kHz. Currents were sampled at 4 kHz by a 12-bit resolution analog-to-digital converter and stored on the hard disk of a computer. All electrophysiological experiments were done at room temperature (22–24°C).

The transient-outward K+ current (Ito) was evoked by 500-ms depolarizing pulses to test potentials between –40 and +60 mV (0.2 Hz). The holding potential was set at –80 mV, and a 100-ms prepulse was applied to –60 mV to inactivate the fast Na+ current. For each test pulse, Ito amplitude was measured as the difference between the peak outward current and the steady-state current level at the end of the depolarizing pulse. All electrophysiological data were normalized as current densities by dividing measured current amplitude by whole cell capacitance.

Enzyme assays. TrxR and Trx activities were measured by the insulin disulfide reduction assay (9). For TrxR, aliquots of cell lysate from tissue samples were added to a reaction mixture containing 80 mmol/l HEPES (pH 7.5), 6 mmol/l EDTA, 0.9 mg/ml NADPH, and 2 mg/ml insulin. The reaction was started by adding 5 µmol/l Escherichia coli Trx, and the samples were heated to 37°C for 20 min. The reaction was stopped by adding 500 µl of 0.4 mg/ml DTNB plus 6 mol/l guanidine-HCl in 0.2 mol/l Tris·HCl (pH 8.0), and the absorbance was read at 412 nm in a spectrophotometer (ThermoSpectronic, Waltham, MA). The measured absorbance from samples was compared with the standard curves generated with known amounts of rat liver TrxR. For total Trx activity, aliquots of extracts from tissue samples were added to a reaction mixture containing 47 mmol/l KH2PO4 buffer, 0.1 mmol/l EDTA, 0.2 mmol/l NADPH, and 0.5 mg insulin. The reaction was started by adding 1 unit bovine TrxR, and the absorbance was read at 340 nm for 5 min at 37°C. Measured activity was expressed in milliunits per milligram protein with 1 mU Trx activity defined as 1 nmol NADPH oxidized per minute.

GR and Grx activities were also measured by standard spectrophotometric techniques (4, 25). To assay GR, tissue samples were homogenized in ice-cold Tris buffer (0.1 mol/l, pH 8.0, with 2 mmol/l EDTA) and centrifuged at 4°C (6,000 g) for 30 min. A 200-µl aliquot of supernatant was added to a cuvette containing KH2PO4 buffer (0.2 mol/l, pH 7.0) plus 2 mmol/l EDTA, 20 mmol/l GSSG, and 2 mmol/l NADPH. The change in absorbance at 340 nm was monitored for 5 min at 30°C. GR activity was expressed in milliunits and defined as the amount of enzyme catalyzing the reduction of 1 nmol NADPH per minute. For Grx activity, aliquots of cell lysate from tissue samples were added to a reaction mixture containing 0.2 mmol/l NADPH, 0.5 mmol/l GSH, 0.1 mol/l KH2PO4 buffer (pH 7.4), and 0.4 units GR. The reaction was initiated by adding 2 mmol/l hydroxy ethyl disulfide, and the NADPH absorbance at 340 nm was monitored over 5 min. Activity was expressed in milliunits per milligram protein with 1 mU Grx activity defined as 1 nmol NADPH oxidized per minute.

Western blot analysis and real-time PCR. Protein extracts of isolated cells or tissue samples were immunoblotted for target proteins following standard protocols. Samples of total protein from isolated myocytes were analyzed to quantify the abundance of {alpha}-subunits underlying Ito, Kv4.2 and Kv4.3, plus the channel accessory subunit KChIP2 that controls the surface expression of Kv4.x {alpha}-subunits (20). The specificity of changes in Kv4.x protein level post-MI was assessed by comparing data with the Kv1.5 {alpha}-subunit, which contributes to delayed rectifier current in rat myocytes (20). Cytosolic protein from tissue samples was immunoblotted for Trx, TrxR, and Txnip. Briefly, the samples were prepared by homogenizing tissues or lysing cells in 1x radioimmunoprecipitation assay buffer (PBS containing 1% NP-40, 0.5% deoxycholate, and 0.1% SDS, pH 7.4). The protein concentration was assayed by a kit (Pierce, Rockford, IL), and equal amounts of protein from all samples were resolved in 10% polyacrylamide gel, transferred to polyvinylidene difluoride membrane, and blocked with 5% milk in Tris-buffered saline, pH 7.0 containing 0.05% Tween 20. The membranes were probed with primary antibodies against Kv4.2, Kv4.3 (Alamone, Jerusalem, Israel), Kv1.5 (Sigma-Aldrich), KChIP2, TrxR, Trx (Santa Cruz Biotechnology, Santa Cruz, CA), or Txnip (MBL, Nagoya, Japan). GAPDH (Santa Cruz) was used for all analyses as an internal control. Signals were generated using corresponding host-specific second antibody-peroxidase conjugate and chemiluminescence substrate (Pierce). The light emanating from specific protein bands was digitized using an UVP bioimager, and data were expressed as optical density. The antibody used to detect KChIP2 usually gave two bands that showed similar changes after MI. For purposes of quantification, the more prominent lower band was used for densitometry measurements, but the exact nature of these bands is not known. It is possible they represent modified forms of KChIP2 or proteolytic fragments.

For real-time PCR, total RNA was extracted from tissue samples or isolated myocytes using an RNAeasy mini kit (Qiagen, Valencia, CA) following the manufacturer's protocol. cDNA was generated from 1 µg total RNA using random hexamer primers (TaqMan reverse transcription reagents), and 1 µl of the resulting cDNA was PCR amplified for 50 cycles using TaqMan gene expression assays for Kv4.2 (Rn00581940_m1), Kv4.3 (Rn00709608_m1), KChIP2 (Rn01411450_g1), Trx (Rn00587437_m1), TrxR (Rn01503799_m1), Txnip (Rn01533890_g1), and β-actin (rat ACTB). All reverse transcription reagents and gene expression assays were purchased from Applied Biosystems (Foster City, CA). Real-time PCR was performed on duplicate samples using a DNA Engine Opticon 3 System (MJ Research, Alameda, CA). For quantification, the target gene expression relative to β-actin was determined, and data was expressed relative to the sham-operated control samples using the following equation: R = 2Formula, where Ct is the number of cycles needed to achieve a preset threshold value of fluorescence.

Statistical analysis. All results are expressed as means ± SE. The comparisons of two groups were made using a Student's t-test, whereas the comparisons of more than two groups were made by ANOVA. When a significant difference between groups was indicated by the initial analysis, individual paired comparisons were made using a Student-Newman-Keuls t-test. Differences were considered significant at P < 0.05.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Trx system in post-MI rat heart. We have previously shown in rats with chemically induced Type 1 diabetes that cardiac oxidoreductase systems undergo marked changes that are consistent with chronic oxidative stress and increased oxidative modification of cellular proteins (15). In this experimental model, we also found evidence suggesting that the Trx system is involved in regulating the density of Ito in ventricular myocytes (15). Thus, to examine the status of the cardiac Trx system after MI, tissue samples from the left ventricle were assayed for TrxR and Trx activity. Figure 1A shows that when compared with that of the sham-operated control (white bar), TrxR activity was significantly decreased by 32% in post-MI hearts. A second series of assays comparing the activity of Trx showed that in contrast to TrxR, the total Trx activity was significantly increased by 51% in the post-MI group of rats (Fig. 1B). Moreover, parallel experiments examining the status of the Grx system revealed that GR activity in post-MI hearts was significantly decreased by 31% (Fig. 1C), whereas Grx activity was not different between groups (Fig. 1D). It should be noted that in previous studies we have shown that the GSH-to-GSSG ratio is markedly decreased in the left ventricle of post-MI hearts compared with sham-operated controls (28), which is consistent with decreased GR activity.


Figure 1
View larger version (14K):
[in this window]
[in a new window]

  		Fig. 1. Thioredoxin-1 (Trx) and glutaredoxin-1 (Grx) systems in post-myocardial infarction (MI) heart. Tissue samples from left ventricle of sham-operated control (white bars) and post-MI rat hearts were assayed for Trx reductase-1 (TrxR; A), Trx (B), glutathione reductase (GR; C), and Grx (D) activity. Number in parentheses represents number of hearts assayed. *P < 0.05 compared with sham.

 
To explore the basis for changes in activity of the Trx system in greater detail, Western blot and real-time PCR analyses were done in tissue extracts. Figure 2A compares immunoblots of TrxR and Trx in samples obtained from the left ventricle of post-MI and sham-operated hearts. In agreement with measured activities (Fig. 1), the amount of TrxR protein in post-MI hearts was less than that in sham-operated controls, whereas the protein level of Trx was greater in the post-MI group. Mean data from these analyses are summarized in Fig. 2B, which illustrates that the protein abundance of TrxR in post-MI hearts was 46% less than sham-operated hearts, whereas Trx protein was ~3.7-fold higher in the post-MI group (Fig. 2B, middle). The protein level of Txnip, the endogenous negative regulator of Trx (34, 38), was also significantly decreased in post-MI hearts by 17% (Fig. 2B, right). In agreement with changes in protein abundance, mRNA expression of TrxR measured by real-time PCR was decreased in post-MI hearts (Fig. 2C, left), but Trx mRNA levels were not significantly different between groups (Fig. 2C, middle). Finally, as shown in Fig. 2C, right, there was a profound decrease in Txnip expression in post-MI hearts relative to sham-operated hearts.


Figure 2
View larger version (19K):
[in this window]
[in a new window]

  		Fig. 2. Protein abundance and transcript expression of the Trx system. A: immunoblots of TrxR and Trx from post-MI and sham-operated rat hearts are shown along with GAPDH as loading control. B: protein bands corresponding to TrxR (left), Trx (middle), and thioredoxin-interacting protein (Txnip) (right) were measured by densitometry and expressed relative to GAPDH. C: transcript expression of TrxR (left), Trx (middle), and Txnip (right) relative to sham was measured by real-time PCR. Number in parentheses represents number of hearts assayed. *P < 0.05 compared with sham.

 
Redox regulation of K+ channel expression by the Trx system. It is well known from our previous studies (15, 26–28) and others (1, 22) that the basal density of Ito in myocytes from remodeled hearts is downregulated compared with control hearts. We have also shown that this electrophysiological phenotype is reversed by receptor-dependent and -independent agonists that stimulate glucose utilization by the pentose pathway (15, 26–28, 36). In the present study, we used this paradigm of reverse remodeling as an experimental approach to explore the role of the Trx system in regulating K+ channel expression. In particular, we incubated isolated myocytes with a supraphysiological concentration of pyruvate for 4 to 5 h, which we have previously shown upregulates Ito density in myocytes from post-MI hearts (26). This response is illustrated in Fig. 3A, which compares raw current traces from an untreated post-MI myocyte and another treated with 5 mmol/l pyruvate (Fig. 3A, bottom). In the treated myocyte, there is a marked increase in Ito density compared with the time-matched, untreated cell. When examined in several cells (Fig. 3B), pyruvate increased mean Ito density at +60 mV by 71% to a level that was near sham-operated control (post-MI + pyruvate, 25.8 ± 2.0 pA/pF, n = 7; and sham, 31.3 ± 2.6 pA/pF, n = 10; P > 0.05), whereas it had no significant effect on Ito in myocytes from sham-operated hearts treated for the same duration (Fig. 3C). Moreover, the upregulation of Ito by pyruvate was blocked by inhibitors of the pentose pathway (data not shown), which supports the key role of glucose-derived NADPH in redox reactions (10, 28).


Figure 3
View larger version (8K):
[in this window]
[in a new window]

  		Fig. 3. Thioredoxin-mediated regulation of transient-outward K+ current (Ito). A: representative current traces are shown for myocytes from post-MI hearts untreated or treated with 5 mmol/l pyruvate (Pyr) for 4 to 5 h. B: myocytes from post-MI hearts were treated with 5 mmol/l Pyr for 4 to 5 h with or without auranofin (AF; 10 nmol/l) or 13-cis-retinoic acid (RA; 1 µmol/l). C: myocytes from sham-operated hearts were treated with 5 mmol/l Pyr for 4 to 5 h ({blacktriangleup}). Number in parentheses represents number of myocytes examined from 3–4 post-MI rats. Vm, membrane voltage. *P < 0.05 compared with untreated myocytes (bullet).

 
To probe the role of the Trx system in controlling K+ channel remodeling, the pyruvate protocol was repeated after pretreating myocytes for 30 min with one of two specific inhibitors of TrxR: auranofin (AF; 10 nmol/l) or 13-cis-retinoic acid (RA; 1 µmol/l) (15, 24). As shown in Fig. 3B (white circles and squares), both compounds blocked the effect of pyruvate to increase Ito density in myocytes from post-MI hearts. Neither blocker alone altered Ito density (+60 mV) in post-MI myocytes treated for 4 to 5 h (RA treated, 17.2 ± 1.8 pA/pF, n = 11; AF treated, 18.6 ± 2.2 pA/pF, n = 7; and untreated, 15.1 ± 1.5 pA/pF, n = 18; P > 0.05) nor sham-operated control myocytes treated for the same duration (RA treated, 29.9 ± 3.6 pA/pF, n = 10; AF treated, 32.2 ± 4.1 pA/pF, n = 8; and untreated, 31.3 ± 2.6 pA/pF, n = 10; P > 0.05). It should also be noted that AF and RA pretreatment of post-MI myocytes blocked the electrophysiological effect of dichloroacetate (1.5 mmol/l; data not shown), which upregulates Ito density similar to pyruvate (28).

The molecular mechanisms of K+ channel remodeling in the post-MI heart have been studied by several laboratories that have concluded that the downregulation of transcript expression plays a significant role in the decreased K+ currents characteristic of the failing heart (3, 22). Thus, to determine whether pyruvate treatment in our experiments affected the expression of K+ channels in a redox-sensitive manner, mRNA levels of channel transcripts were quantified by real-time PCR in isolated myocyte suspensions from post-MI and sham-operated hearts. Figure 4A shows that relative to sham-operated control, mRNA levels of Kv4.2, Kv4.3, and KChIP2 were markedly less in myocytes from post-MI hearts. When these myocytes were treated with 5 mmol/l pyruvate for 4 to 5 h, the transcripts for Kv4.2, Kv4.3, and KChIP2 were significantly increased compared with time-matched, untreated myocytes from the same group (Fig. 4B). The pyruvate-stimulated upregulation of Kv4.2 and Kv4.3 mRNA was blocked when myocytes were pretreated for 30 min with 10 nmol/l AF, whereas this inhibitor did not change the pyruvate response of KChIP2. In comparison, none of the channel transcripts studied was significantly altered by pyruvate (4 to 5 h) in myocytes from sham-operated hearts (Fig. 4C).


Figure 4
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 4. Upregulation of K+ channel expression by Pyr. A: mRNA levels of Kv4.2, Kv4.3, and KChIP2 in isolated myocytes from post-MI (black bars) and sham-operated hearts were measured by real-time PCR. Mean data for the post-MI group are expressed relative to sham. *P < 0.05 compared with sham. B: myocytes from post-MI hearts were treated with 5 mmol/l Pyr in the absence or presence of 10 nmol/l AF. Mean data are expressed relative to untreated myocytes from post-MI hearts. *P < 0.05 compared with untreated myocytes. C: myocytes from sham-operated hearts were treated with Pyr for 4 to 5 h. Mean data were derived from 4–6 sham-operated and post-MI hearts.

 
In parallel with real-time PCR studies, Western blots were also analyzed in myocyte suspensions from post-MI and sham-operated hearts treated with pyruvate. Figure 5A compares immunoblots of channel protein in representative samples from sham-operated and post-MI hearts, showing that protein abundance of Kv4.2, Kv4.2, and KChIP2 subunits was decreased in myocytes from the post-MI group but that Kv1.5 was unchanged. Figure 5B compares mean data from several hearts and shows that the levels of Kv4.2, Kv4.3, and KChIP2 protein were ~40% less in post-MI hearts compared with sham-operated hearts, whereas Kv1.5 was not different between groups. As summarized in Fig. 5C, pyruvate treatment of myocytes from post-MI hearts significantly increased Kv4.2 (left) and Kv4.3 (middle) protein abundance relative to the time-matched, untreated myocytes, and this response was blocked by AF. KChIP2 protein levels also significantly increased after pyruvate treatment, but AF did not block this response. In contrast to findings in post-MI myocytes, pyruvate did not significantly affect the protein abundance of channel subunits in myocytes from sham-operated hearts (Fig. 5D). These data suggest, along with findings summarized in Fig. 4, that the Trx system regulates mRNA expression and protein abundance of Kv4.x {alpha}-subunits but not the accessory protein KChIP2.


Figure 5
View larger version (20K):
[in this window]
[in a new window]

  		Fig. 5. Upregulation of K+ channel protein by Pyr. A: representative examples of Western blots of Kv4.2, Kv4.3, KChIP2, and Kv1.5 protein in suspensions of isolated myocytes from post-MI and sham-operated hearts. B: mean densitometric measurements of channel subunits are expressed relative to GAPDH. *P < 0.05 compared with sham. C: myocytes from post-MI hearts were treated for 4 to 5 h with 5 mmol/l Pyr without and with 10 nmol/l AF. *P < 0.05 compared with untreated myocytes. D: myocytes from sham-operated hearts were pretreated with 5 mmol/l Pyr for 4 to 5 h. Mean data shown in B–D were derived from 4–6 sham-operated and post-MI hearts.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Redox regulation by oxidoreductase systems. Electrical remodeling in the post-MI heart is postulated to involve oxidative stress resulting from increased production of ROS and/or deficits in antioxidant defenses (8, 10, 31). The functional impact of excess ROS levels in cardiac myocytes includes changes in the redox state of cellular proteins, some of which can be reversed. In particular, reversible protein oxidation mainly involves the free sulfhydryl (–SH) side chain of cysteine residues that can undergo a number of different molecular modifications which can elicit positive or negative changes in protein function (23). The redox status of cellular proteins is maintained in large part by ubiquitous, thiol disulfide oxidoreductase networks such as the Trx and Grx systems. The primary function of these systems in the cytoplasm and mitochondria is to convert oxidized sulfhydryl groups to their normally reduced state, although they also participate in antioxidant reactions (7, 14, 24, 25, 37). Furthermore, it should be noted that the thio-ether side chain of methionine residues can also undergo oxidation to methionine sulfoxide, which is reduced by the action of specific reductases that require reduced Trx as cofactor (32). Thus, under conditions in which oxidoreductase systems are impaired or ROS levels are markedly elevated, there is a net increase in oxidized proteins that can dramatically affect the physiological function of cells.

The expression of Trx and Grx in many cell types is sensitive to stress factors such as inflammation and excess ROS generation (1, 19, 37). It is proposed that stress-induced upregulation of oxidoreductase activity is an important cellular compensatory mechanism protecting proteins from irreversible oxidative damage. This mechanism has not been well characterized in heart, but recent studies of myocarditis in rodent models (19, 30) and in human heart (21) have shown that Trx expression is increased during the immune response. In our study, done 6–8 wk post-MI, total Trx activity (Fig. 1B) and protein abundance (Fig. 2B) in the left ventricle were significantly greater than in sham-operated controls, which is consistent with (oxidative) stress-induced upregulation. A qualitatively similar increase in Trx activity was also observed in the rat heart after 3–5 wk of experimental diabetes (15). However, the increase in overall Trx status post-MI was not accompanied by an increase in Trx mRNA (Fig. 2C) but rather by a decrease in mRNA and protein abundance of the endogenous Trx inhibitor, Txnip (Fig. 2, B and C). The opposite changes in Txnip expression and Trx activity are consistent with studies in mouse ventricle with pressure-overload hypertrophy (38) and in cultured rat ventricular myocytes exposed to mechanical or oxidative stress (34) and implicate Txnip as an important regulator of Trx function along with TrxR. However, it should be noted that cytosolic levels of Trx may be affected by the direct binding with other endogenous proteins or by the translocation to and from the nucleus (2, 24, 37, 38), which may be regulated by Txnip (2).

In contrast to changes in Trx, the activity, protein abundance, and mRNA levels of TrxR were significantly decreased in post-MI hearts (Figs. 1A and 2). Since the amount of active (reduced) Trx depends on the activity of TrxR, the inhibition of the latter in the post-MI heart should result in correspondingly decreased levels of active Trx. In this regard, it should be noted that the Trx assay used in our studies measures total activity and does not assess the amount of enzyme that is in the inactive (oxidized) form. Nevertheless, inhibited TrxR has many effects on cellular function linked to suppressed Trx activity, but the pathophysiological signals in the heart mediating the downregulation of TrxR are not known. Some ROS, electrophilic prostaglandin derivatives, and lipid aldehydes rapidly inhibit TrxR and can subsequently lead to increased mRNA levels (29), although in some cell types, mRNA levels are decreased (29). Hence, it is possible that soon after MI in our model, TrxR expression was increased as a compensatory response, but our experiments indicate that TrxR under chronic pathological conditions is markedly suppressed.

The reducing activities of the Trx and Grx systems both require metabolically derived NADPH, which is generated mainly by the cytosolic pentose pathway (10). The rate-limiting enzyme of this pathway, glucose-6-phosphate dehydrogenase (G6PD), has been shown to have a significant impact on myocyte redox state and function (10). Although the link between glucose utilization and cell redox state is not completely understood, mitochondrial enzymes and metabolites appear to play an important role, such as those related to pyruvate dehydrogenase. In particular, exogenous pyruvate increases pyruvate carboxylation (17, 18) and activates pyruvate dehydrogenase (17). The oxidoreductase-related effects of pyruvate are proposed to be mediated by an increase in mitochondrial citrate formation that effluxes to the cytoplasm to inhibit phosphofructokinase, thus diverting glucose-6-phosphate into the pentose pathway and increasing NADPH bioavailability (18). In support of this mechanism, we found in the present study that pyruvate-induced upregulation of Ito density in myocytes from post-MI hearts was blocked by inhibitors of G6PD, which is similar to what we reported previously using dichloroacetate (28). This finding as well as other data (17, 18, 26, 27, 36) indicate that components of glucose metabolism have essential cellular functions associated with redox regulation that are independent of ATP formation.

Control of K+ channel expression by the Trx system. In our study we used the upregulation of Ito density by exogenous pyruvate as a means to explore the role of the Trx system in regulating K+ channel expression. Since specific pharmacological inhibitors of Trx are not readily available, we focused our attention on its upstream regulator, TrxR. This enzyme is a ~110–120-kDa homodimer containing a unique COOH-terminal selenocysteine residue that is essential for catalytic activity (9, 24, 37). The functional selenocysteine of TrxR makes this enzyme, as well as other selenoenzymes, sensitive to inhibition by gold-containing compounds such as aurothioglucose and AF (24). In the present study, we tested AF and RA based on their selectivity for TrxR (15, 24), and our results (Fig. 3B) suggest that the Trx system plays a key role in the redox regulation of K+ channels underlying Ito, although we cannot completely rule out a role for the Grx system (7, 14, 25), which was also significantly altered by chronic MI. Figure 4 further suggests that the regulation by the Trx system occurs at least at the level of transcription, since pyruvate increased mRNA levels of Kv4.2 and Kv4.3 in isolated myocytes in an AF-sensitive manner. Moreover, this increase in mRNA was translated into increased amount of channel {alpha}-subunits (Fig. 5). Thus our data are in agreement with previous studies showing that changes in {alpha}-subunit abundance and ionic currents in the remodeled heart parallel alterations in mRNA expression (3, 22). It should also be pointed out that the pyruvate-induced increase in mRNA and protein expression of KChIP2 was not inhibited by AF, suggesting that the Trx system is not involved in regulating this accessory subunit. The mechanisms underlying the regulation of KChIP2 in the remodeled ventricle are unclear, but it is possible that the Grx system may be involved. We also cannot exclude the involvement of Kvβ accessory subunits, which have been shown to regulate surface expression of Kv4.3 {alpha}-subunits (1).

Although our data show quantitatively similar decreases in {alpha}-subunit abundance, mRNA expression, and Ito density in myocytes from post-MI hearts under basal conditions, the upregulation of subunit mRNA and protein by pyruvate was relatively modest compared with the increase in Ito. This may be due to our use of myocytes from different regions of the left ventricle and septum or to in vitro differences in the metabolic sensitivity of myocytes to pyruvate. However, it is also possible that the overall response of Ito to pyruvate in post-MI myocytes includes the direct redox modification of channel subunits or regulatory proteins. Indeed, recent data from our laboratory suggest that Kv currents acutely inhibited by exogenous oxidants in normal ventricular myocytes are normalized by oxidoreductase systems but that this regulation is mediated by changes in tyrosine kinase or phosphatase pathways (16). Hence, the overall increase in Ito density elicited by pyruvate in myocytes from post-MI hearts may reflect a combination of redox-sensitive transcriptional and posttranslational mechanisms.

The cellular targets of the Trx system that regulate the expression of cardiac K+ channel subunits are currently unknown. On one hand, it may be postulated that Trx directly controls the binding activity of transcription factors that normally regulate K+ channel subunit expression (12). Alternatively, the reduced form of Trx may normally inhibit transcriptional or translational repressors of channel subunits (5), such that repressor activity is enhanced when the Trx system is impaired, as in the post-MI heart. The Trx system may also regulate the redox state of signaling pathways that are upstream of transcriptional events, such as JAK-STAT (6), JNK, or MEK-ERK pathways (13). Lastly, it is possible that redox mechanisms participate in controlling the stability of subunit mRNA (39). Given the complexities of homeostatic regulation of ion channel expression, it is clear that additional experimentation is warranted to identify the specific redox-sensitive steps involved in regulating Kv channel expression.

Limitations. Although our study implicates the Trx system as an important regulator of K+ channel remodeling, certain limitations must be kept in mind. First, we did not fully characterize the impact of chronic MI on the intracellular redox state of myocytes. We have characterized the status of oxidoreductase systems post-MI and previously showed a decrease in intracellular GSH-to-GSSG ratio (28), but we have not measured the extent or types of protein oxidation in the remodeled ventricle, the activity of G6PD, or the bioavailability of NADPH. Second, to maximize the cellular material needed for Western blot analysis and real-time PCR studies, we used isolated myocytes from the left ventricle and septum and did not examine regional differences. It is well known that regional and transmural differences in the expression of certain Kv channels exist in normal rat heart (20) and that these differences are diminished during the remodeling process. It is also possible that oxidoreductase systems and the metabolic pathways that support them show regional differences that change in unique ways following MI. Certainly, additional studies are needed to specifically correlate biochemical with electrophysiological properties based on cardiac region. Finally, the expression of certain Kv channels differs between species (20), such that the electrophysiological functions of the Trx and Grx systems would need to be confirmed in larger mammals.

In conclusion, the expression of Kv4.x channels underlying Ito is regulated in a redox-sensitive manner by the Trx system, which is impaired in the post-MI heart. The sensitivity of ion channel expression to oxidative stress-induced changes in cell redox state suggests that endogenous Trx and Grx systems may be effective therapeutic targets to limit or reverse pathogenic electrical remodeling of the failing heart.


   GRANTS
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by a National Heart, Lung, and Blood Institute Grant HL-66446.


   FOOTNOTES
 

Address for reprint requests and other correspondence: G. J. Rozanski, Dept. of Cellular and Integrative Physiology, Univ. of Nebraska Medical Ctr., 985850 Nebraska Medical Ctr., Omaha, NE 68198-5850 (e-mail: grozansk{at}unmc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


   REFERENCES
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Aimond F, Kwak SP, Rhodes K, Nerbonne JM. Accessory Kvβ1 subunits differentially modulate the functional expression of voltage-gated K+ channels in mouse ventricular myocytes. Circ Res 96: 451–458, 2005.[Abstract/Free Full Text]
  2. Ago T, Sadoshima J. Thioredoxin and ventricular remodeling. J Mol Cell Cardiol 41: 762–773, 2006.[CrossRef][Web of Science][Medline]
  3. Armoundas AA, Wu R, Juang G, Marban E, Tomaselli GF. Electrical and structural remodeling of the failing ventricle. Pharmacol Ther 92: 213–230, 2001.[CrossRef][Web of Science][Medline]
  4. Carlberg I, Mannervik B. Glutathione reductase. Methods Enzymol 113: 484–490, 1985.[Web of Science][Medline]
  5. Costantini DL, Arruda EP, Agarwal P, Kim KH, Zhu Y, Zhu W, Lebel M, Cheng CW, Park CY, Pierce SA, Guerchicoff A, Pollevick GD, Chan TY, Kabir MG, Cheng SH, Husain M, Antzelevitch C, Srivastava D, Gross GJ, Hui CC, Backx PH, Bruneau BG. The homeodomain transcription factor Irx5 establishes the mouse cardiac ventricular repolarization gradient. Cell 123: 347–358, 2005.[CrossRef][Web of Science][Medline]
  6. El-Adawi Deng L, Tramontano A, Smith S, Mascareno E, Ganguly K, Casillo R, El-Sherif N. The functional role of the JAK-STAT pathway in post-infarction remodeling. Cardiovasc Res 57: 129–138, 2003.[Abstract/Free Full Text]
  7. Fernanades AP, Holmgren A. Glutaredoxins: glutathione-dependent redox enzymes with functions far beyond a simple thioredoxin backup system. Antioxid Redox Signal 6: 63–74, 2004.[CrossRef][Web of Science][Medline]
  8. Hess ML, Kukreja RC. Free radicals, calcium homeostasis, heat shock proteins, and myocardial stunning. Ann Thorac Surg 60: 760–766, 1995.[Abstract/Free Full Text]
  9. Holmgren A, Björnstedt M. Thioredoxin and thioredoxin reductase. Methods Enzymol 252: 199–208, 1995.[CrossRef][Web of Science][Medline]
  10. Jain M, Cui L, Brenner DA, Wang B, Handy DE, Leopold JA, Loscalzo J, Apstein CS, Liao R. Increased myocardial dysfunction after ischemia-reperfusion in mice lacking glucose-6-phosphate dehydrogenase. Circulation 109: 898–903, 2004.[Abstract/Free Full Text]
  11. Jeroudi MO, Hartley CJ, Bolli R. Myocardial reperfusion injury: role of oxygen free radicals and potential therapy with antioxidants. Am J Cardiol 73: 2B–7B, 1994.[CrossRef][Medline]
  12. Jia Y, Takimoto K. GATA and FOG2 transcription factors differentially regulate the promoter for Kv4.2 K+ channel gene in cardiac myocytes and PC12 cells. Cardiovasc Res 60: 278–287, 2003.[Abstract/Free Full Text]
  13. Jia Y, Takimoto K. Mitogen-activated protein kinases control KChIP2 gene expression. Circ Res 98: 386–393, 2006.[Abstract/Free Full Text]
  14. Jung CH, Thomas JA. S-glutathiolated hepatocyte proteins and insulin disulfides as substrates for reduction by glutaredoxin, thioredoxin, protein disulfide isomerase, and glutathione. Arch Biochem Biophys 335: 61–72, 1996.[CrossRef][Web of Science][Medline]
  15. Li X, Xu Z, Li S, Rozanski GJ. Redox regulation of Ito remodeling in diabetic rat heart. Am J Physiol Heart Circ Physiol 288: H1417–H1424, 2005.[Abstract/Free Full Text]
  16. Liang H, Li X, Li S, Zheng MQ, Rozanski GJ. Oxidoreductase regulation of Kv currents in rat ventricle. J Mol Cell Cardiol. 44: 1062–1071, 2008.[CrossRef][Medline]
  17. Mallet RT. Pyruvate: metabolic protector of cardiac performance. Proc Soc Exp Biol Med 223: 136–148, 2000.[Abstract/Free Full Text]
  18. Mallet RT, Sun J. Antioxidant properties of myocardial fuels. Mol Cell Biochem 253: 103–111, 2003.[CrossRef][Web of Science][Medline]
  19. Miyamoto M, Kishimoto C, Shioji K, Nakamura H, Toyokumi S, Nakayama Y, Kita M, Yodoi J, Sasayama S. Difference in thioredoxin expression in viral myocarditis in inbred strains of mice. Jpn Circ J 65: 561–564, 2001.[CrossRef][Medline]
  20. Nerbonne JM, Kass RS. Physiology and molecular biology of ion channels contributing to ventricular repolarization. In: Cardiac Repolarization: Bridging Basic and Clinical Science, edited by Gussak I, Antzelevitch C, Hammill SC, Shen WK, and Bjerregaard P. Totowa, NJ: Humana, 2003, p. 25–62.
  21. Nimata M, Kishimoto C, Shioji K, Ishizaki K, Kitaguchi S, Hashimoto T, Nagata N, Kawai C. Upregulation of redox-regulating protein, thioredoxin, in endomyocardial biopsy samples of patients with myocarditis and cardiomyopathies. Mol Cell Biochem 248: 193–196, 2003.[CrossRef][Web of Science][Medline]
  22. Oudit GV, Kassiri Z, Sah R, Ramirez RJ, Zobel C, Backx PH. The molecular physiology of the cardiac transient outward potassium current (Ito) in normal and diseased myocardium. J Mol Cell Cardiol 33: 851–872, 2001.[CrossRef][Web of Science][Medline]
  23. Paget MS, Buttner MJ. Thiol-based regulatory switches. Annu Rev Genet 37: 91–121, 2003.[CrossRef][Web of Science][Medline]
  24. Powis G, Mustacichi D, Coon A. The role of the redox protein thioredoxin in cell growth and cancer. Free Radic Biol Med 29: 312–322, 2000.[CrossRef][Web of Science][Medline]
  25. Raghavachari N, Lou MF. Evidence for the presence of thioltransferase in the lens. Exp Eye Res 63: 433–441, 1996.[CrossRef][Web of Science][Medline]
  26. Rozanski GJ, Xu Z, Zhang K, Patel KP. Altered K+ current of ventricular myocytes in rats with chronic myocardial infarction. Am J Physiol Heart Circ Physiol 274: H259–H265, 1998.[Abstract/Free Full Text]
  27. Rozanski GJ, Xu Z. A metabolic mechanism for cardiac K+ channel remodeling. Clin Exp Pharmacol Physiol 29: 132–137, 2002.[CrossRef][Web of Science][Medline]
  28. Rozanski GJ, Xu Z. Glutahtione and K+ channel remodeling in postinfarction rat heart. Am J Physiol Heart Circ Physiol 282: H2346–H2355, 2002.[Abstract/Free Full Text]
  29. Rundlöf AK, Arnér ES. Regulation of the mammalian selenoprotein thioredoxin reductase 1 in relation to cellular phenotype, growth, and signaling events. Antioxid Redox Signal 6: 41–52, 2004.[CrossRef][Web of Science][Medline]
  30. Shioji K, Kishimoto C, Nakamura H, Toyokuni S, Nakayama Y, Yodoi J, Sasayama S. Upregulation of thioredoxin (TRX) expression in giant cells myocarditis in rats. FEBS Lett 472: 109–113, 2000.[CrossRef][Web of Science][Medline]
  31. Singal PK, Khaper N, Palance V, Kumar D. The role of oxidative stress in the genesis of heart disease. Cardiovasc Res 40: 426–432, 1998.[Abstract/Free Full Text]
  32. Stadtman ER. Protein oxidation and aging. Free Radic Res 40: 1250–1258, 2006.[CrossRef][Web of Science][Medline]
  33. Takimoto K, Yang EK, Conforti L. Palmitoylation of KChIP splicing variants is required for efficient cell surface expression of Kv4.3 channels. J Biol Chem 277: 26904–26911, 2002.[Abstract/Free Full Text]
  34. Wang Y, De Keulenaer GW, Lee RT. Vitamin D3-up-regulated protein-1 is a stress-responsive gene that regulates cardiomyocyte viability through interaction with thioredoxin. J Biol Chem 277: 26496–26500, 2002.[Abstract/Free Full Text]
  35. Wickenden AD, Kaprielian R, Kassiri Z, Tsoporis JN, Tsushima R, Fishman GI, Backx PH. The role of action potential prolongation and altered intracellular calcium handling in the pathogenesis of heart failure. Cardiovasc Res 37: 312–323, 1998.[Abstract/Free Full Text]
  36. Xu Z, Patel KP, Lou MF, Rozanski GJ. Up-regulation of K+ channels in diabetic rat ventricular myocytes by insulin and glutathione. Cardiovasc Res 53: 80–88, 2002.[Abstract/Free Full Text]
  37. Yamawaki H, Haendeler J, Berk BC. Thioredoxin: a key regulator of cardiovascular homeostasis. Circ Res 93: 1029–1033, 2003.[Abstract/Free Full Text]
  38. Yoshioka J, Schulze PC, Cupesi M, Sylvan JD, MacGillivray C, Gannon J, Huang H, Lee RT. Thioredoxin-interacting protein controls cardiac hypertrophy through regulation of thioredoxin activity. Circulation 109: 2581–2586, 2004.[Abstract/Free Full Text]
  39. Zhang TT, Takimoto K, Stewart AF, Zhu C, Levitan ES. Independent regulation of cardiac Kv4.3 potassium channel expression by angiotensin II and phenylephrine. Circ Res 88: 476–482, 2001.[Abstract/Free Full Text]



This article has been cited by other articles:


Home page
 Am. J. Physiol. Cell Physiol.Home page
M.-Q. Zheng, K. Tang, M. C. Zimmerman, L. Liu, B. Xie, and G. J. Rozanski
Role of {gamma}-glutamyl transpeptidase in redox regulation of K+ channel remodeling in postmyocardial infarction rat hearts
Am J Physiol Cell Physiol, August 1, 2009; 297(2): C253 - C262.
[Abstract] [Full Text] [PDF]

Home page
 Exp PhysiolHome page
S. Li, M.-Q. Zheng, and G. J. Rozanski
Glutathione homeostasis in ventricular myocytes from rat hearts with chronic myocardial infarction
Exp Physiol, July 1, 2009; 94(7): 815 - 824.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
295/1/H416    most recent
91446.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (1)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Li, X.
Right arrow Articles by Rozanski, G. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Li, X.
Right arrow Articles by Rozanski, G. J.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2008 by the American Physiological Society.