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Am J Physiol Heart Circ Physiol 293: H238-H245, 2007. First published March 2, 2007; doi:10.1152/ajpheart.01122.2006
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TNF-{alpha} downregulates transient outward potassium current in rat ventricular myocytes through iNOS overexpression and oxidant species generation

María Fernández-Velasco ,* Gema Ruiz-Hurtado,* Olivia Hurtado, Maria Ángeles Moro, and Carmen Delgado

Institute of Pharmacology and Toxicology (Consejo Superior de Investigaciones Científicas-Universidad Complutense Madrid), Universidad Complutense, Madrid, Spain

Submitted 13 October 2006 ; accepted in final form 25 February 2007


   ABSTRACT
TOP
 ABSTRACT
METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Tumor necrosis factor-{alpha} (TNF-{alpha}) is a proinflammatory cytokine that has been implicated in the pathogenesis of heart failure. Prolongation of the action potential duration and downregulation of several K+ currents might participate in the genesis of arrhythmias associated with chronic heart failure. Little information is available related to the mechanism by which TNF-{alpha} modulates cardiac K+ channels. The present study analyzes the effect of TNF-{alpha} on the transient outward K+ current (Ito) in rat ventricular myocytes, using the whole cell patch-clamp technique. We found that TNF-{alpha} is able to induce a significant reduction of Ito density, modifies its inactivation, and downregulates the Kv4.2 protein expression, while calcium current density is not affected. We have also demonstrated that the reduction of Ito density induced by TNF-{alpha} was prevented by the selective inducible nitric oxide synthase (iNOS) inhibitor 1400-W, the protein synthesis inhibitor cycloheximide, the antioxidant tocopherol, and the superoxide dismutase mimetic manganese(III) tetrakis (4-benzoic acid) porphyrin. In addition, a reduced Ito density was recorded in ventricular myocytes exposed to peroxynitrite, supporting a possible participation of this oxidant in the effects of TNF-{alpha} on Ito. We conclude that TNF-{alpha} exposure, through iNOS induction and generation of oxidant species, promotes electrophysiological changes (decreased Ito and action potential duration prolongation) in rat ventricular myocytes, providing new insights into how cytokines modulate K+ channels in the heart.

cardiac electrophysiology; tumor necrosis factor; inducible nitric oxide synthase; oxidant species

TUMOR NECROSIS FACTOR (TNF)-{alpha} is a proinflammatory cytokine with pleiotropic properties that is upregulated in a variety of cardiovascular diseases, such as dilated cardiomyopathy, acute myocarditis, cardiac allograft rejection, myocardial infarction, and congestive heart failure (47). Ventricular myocytes from failing mammalian hearts show prolongation of the action potential duration (APD) and downregulation of several K+ currents, including the transient outward K+ current (Ito) sensitive to 4-aminopyridine (4-AP) (4, 6, 23, 31). The decrease of outward currents and the prolongation of APD cause a predisposition to suffering triggered activity and may contribute to arrhythmogenesis in heart failure (43). In this context, transgenic mice with cardiac-specific overexpression of TNF-{alpha} develop cardiac hypertrophy and congestive heart failure (8, 29). Also, ventricular myocytes isolated from these animals show prolonged APDs (35). Cardiac hypertrophy, dilatation, and reduction of Ito are common findings in transgenic mice overexpressing different proteins. However, little information is available analyzing the hypothesis that reduction of Ito could be mediated by TNF-{alpha} (46).

Cardiac hypertrophy and heart failure are associated with an increased production of oxidant species (54). Interestingly, oxidant species have been implicated in the hypertrophic effect induced by TNF-{alpha} in neonatal and adult rat cardiomyocytes (41, 57).

TNF-{alpha} upregulates inducible nitric oxide synthase (iNOS) in cardiac myocytes (1, 16, 26). Induction of iNOS produces larger amounts of nitric oxide (NO). Moreover, underlying inflammation, cells also trigger the formation of superoxide from NADPH oxidase, and this anion may also be formed from the uncoupling of the mitochondrial respiratory chain due to the excessive iNOS-induced NO formation (33). The reaction of NO and superoxide forms peroxynitrite (3). Some studies have suggested that the release of NO and generation of superoxide by the induction of iNOS might contribute to the pathogenesis of cardiovascular diseases (12, 40). Based on this evidence, a possible hypothesis is that TNF-{alpha} could modulate Ito through a mechanism that would involve iNOS upregulation and increased generation of oxidant species, including peroxynitrite, in rat ventricular myocytes. To test this hypothesis, the present study analyzes the effect of TNF-{alpha} on Ito in rat ventricular myocytes and the mechanisms involved.


   METHODS
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 ABSTRACT
 METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Isolation of cardiomyocytes. The animal experimental procedures and care facility were approved by the Bioethical Committee of the Spanish Council for Scientific Research. Apical myocytes were isolated from the hearts of adult male Wistar rats (250–300 g of weight), as previously described (18, 19). Adult male Wistar rats were heparinized (4 IU/g ip) and anesthetized with pentobarbital sodium (50 mg/kg). The hearts were removed and mounted on a Langendorff perfusion apparatus. The ascending aorta was cannulated, and a retrograde perfusion was set up. The hearts were perfused at 36–37°C with a standard calcium-free Tyrode solution containing (in mM) 130 NaCl, 0.4 NaH2PO4, 5.8 NaHCO3, 0.5 MgCl2, 1 CaCl2, 5.4 KCl, 22 glucose, and 25 HEPES, and supplemented with 0.2 mM EGTA (3 min) and then with the same Tyrode solution containing 251 IU/ml of collagenase type II (Worthington) and 0.1 mM CaCl2. After perfusion, the hearts were removed from the Langendorff apparatus. The apical part of the heart was cut off and gently shaken for 3 min in a standard Tyrode solution containing 0.1 mM of CaCl2 to disperse the isolated cells. The resulting cell suspensions were filtered through a 250-µm nylon mesh, centrifuged for 3 min at 20 g, and resuspended in the Tyrode solution containing 0.5 mM CaCl2. Finally, the cells were again centrifuged and resuspended in a store solution containing 1 mM CaCl2.

We used only cells from the apical part of the heart, because our laboratory has previously demonstrated that Ito density was more homogeneous in this region (4). After isolation, cardiomyocytes were incubated for 48 h at 37°C in Tyrode solution (supplemented with 1 mg/ml BSA, 100 IU/ml penicillin, and 0.1 µg/ml streptomycin) (45) in the absence (control) or presence of different drugs.

Patch-clamp experiments. Whole cell configuration of the patch-clamp technique was employed to measure ionic currents and action potentials (APs) (17). Patch-clamp experiments were always performed on control and TNF-{alpha}-treated cells, isolated from the same heart, and recorded on the same day. APs were elicited at 2-s intervals by 1.5-fold excitation threshold current pulses of 2.5 ms in duration. After stabilization of the record, 10 successive APs were recorded. The parameters of the APs for each analyzed cell corresponded to the mean of these 10 APs. The following parameters were measured: AP amplitude and APD at 20, 50, and 90% repolarization.

APs were measured in a standard external solution containing (in mM) 140 NaCl, 4 KCl, 1.1 MgCl2, 1.8 CaCl2, 10 glucose, and 10 HEPES (pH adjusted to 7.4 with NaOH). The recording pipettes contained (in mM) 150 KCl, 1 MgCl2, 10 HEPES, 5 EGTA, 5 Na2ATP, and 10 glucose (pH adjusted to 7.2 with KOH).

The Ito, defined as the 3 mM 4-AP-sensitive current, was measured as described previously (19). Ito was evoked by depolarizing pulses from a holding potential of –80 mV to voltage steps from –30 to +60 mV in 10-mV increments for 500 ms; a prestep to –40 mV was used to inactivate sodium current. Inactivation of Ito was measured by a double protocol: a 2,000-ms conditioning prepulse from a holding potential of –80 mV to potentials between –100 and 0 mV (in 10-mV steps) was followed by a 500-ms depolarizing pulse to +40 mV.

Ito amplitude was taken as the difference between the peak outward current and the current at the end of the pulse. Current density was calculated from the current amplitude normalized by the membrane capacitance (Cm) (14).

The solution for Ito current recordings contained (in mM) 135 NaCl, 10 glucose, 10 HEPES, 1 MgCl2, 1 CaCl2, 5.4 KCl, and 2 CoCl2 (pH adjusted to 7.4 with NaOH). The intracellular recording pipette solution contained (in mM) 125 potassium aspartate, 25 KCl, 10 EGTA, 5 HEPES, 1 MgCl2, 5 Na2ATP, and 0.4 Na2GTP (pH adjusted to 7.2 with KOH).

The L-type calcium current (ICaL) was elicited by 200-ms steps from –40 to +60 mV. To inactivate the Na+ current, cells were predepolarized to –50 mV by a slow (500-ms) voltage ramp for 50 ms before each test depolarization was applied. The solution for ICaL recordings contained (in mM) 135 NaCl, 1 MgCl2, 20 CsCl, 10 glucose, 10 HEPES, and 1.8 CaCl2, with the pH adjusted to 7.4 with CsOH. The intracellular recording pipette solution contained (in mM) 100 CsCl, 20 tetraethylammonium, 5 EGTA, 10 HEPES, 5 Na2ATP, 0.4 Na2GTP, and 5 Na2 creatine phosphate (pH adjusted to 7.2 with CsOH).

Drugs. Recombinant TNF (rat) was purchased from PeproTech EC, tocopherol and cycloheximide (CHX) from Sigma, manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP) and peroxynitrite from Calbiochem, and N-[3-(aminomethyl) benzylacetamidine], 1400-W, was a kind gift of Richard G. Knowles (GSK-UK, Stevenage, UK).

The amount of peroxynitrite in the stock solution was determined spectrophotometrically before each experiment using the reported molar extinction coefficient for peroxynitrite: {varepsilon} = 1,670 mol·l–1·cm–1.

Reverse transcriptase polymerase chain reaction. RNA was isolated from the cardiomyocytes using TRIzol reagent (Invitrogen), followed by chloroform extraction and isopropanol precipitation (14).

cDNA synthesis was carried out with 1 µg of RNA and Moloney murine leukemia virus reverse transcriptase (Invitrogen), in accordance with the manufacturer's instructions.

Rat atrial natriuretic factor (ANF) mRNA was amplified using the following primers: 5'-GGT AGG ATT GAC AGG ATT GGA G-3' (sense), and 5'-CGT GAT AGA TGA AGA CAG GAA G-3' (antisense), leading to 198 base pairs (bp).

Rat c-fos mRNA was amplified using the following primers: 5'-AGT GGT GAA GAC CAT GTC AGG-3' (sense), and 5'-CAT TGG GGA TCT TGC AGG CAG-3' (antisense), leading to 296 bp.

GAPDH was used as an internal standard. GAPDH was amplified using the following primers: 5'-ACC ACA GTC CAT GCC ATC AC-3' (sense), and 5'-TCC ACC ACC CTG TTG CTG TA-3' (antisense), leading to 450 bp. GAPDH was amplified using the same template and in the same PCR where c-fos or ANF was amplified (multiple primers sets in the same tube).

PCR procedure was carried out using the PCR system, MyCycler Thermal Cycler (Bio-Rad), under the following conditions: initial denaturation at 93°C for 3 min, followed by 30 amplification cycles, each consisting of denaturation at 93°C for 30 s, annealing at 57°C for 30 s, and extension at 72°C for 30 s, with an additional extension step at the end of the procedure at 72°C for 5 min. RT-negative controls were obtained for all RT-PCR reactions to exclude genomic contamination (58). The final amount of RT-PCR product for each mRNA species was calculated densitometrically using Quantity One software (Bio Rad) and normalized to GAPDH.

Western blot studies. Cardiomyocytes were homogenized by 10-s sonication at 4°C in homogenization buffer containing protease inhibitors. The amount of protein was quantified by the Bradford method (7). Twenty to forty micrograms were loaded per lane, and the proteins were size separated by 10% SDS polyacrylamide gel electrophoresis. The proteins were blotted onto a polyvinylidene difluoride membrane (Amersham Biosciences), and nonspecific binding sites were blocked overnight at 4°C using 5% dried milk and 0.1% Tween 20 Tris-buffered saline (pH 7.4). Membranes were incubated overnight at 4°C with mouse monoclonal iNOS (1:1,000; BD Transduction Laboratories) or rabbit-polyclonal Kv4.2 (1:500; Alomone) antibodies. A secondary horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG (Amersham Biosciences) was used in combination with the enhanced chemiluminescence detection system (SuperSignal West Pico Chemiluminiscent Substrate, Pierce) to visualize the primary antibodies. Band densities were determined with a laser-scanning densitometer (HP-3970) and Quantity One software (Bio-Rad). Protein loading was controlled using an anti-GAPDH antibody (1:4,000; Ambion). GAPDH was determined for each sample in the same blot where iNOS or Kv4.2 was analyzed. Protein level was normalized to GAPDH.

Statistical analysis. Data are presented as means ± SE. Statistical significance was evaluated by analysis of Student's t-test or ANOVA followed by Bonferroni multiple-comparisons test, when appropriate. Differences with values of P < 0.05 were considered significant.


   RESULTS
TOP
 ABSTRACT
 METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
TNF-{alpha} decreases Ito in rat ventricular myocytes. Initial electrophysiological experiments were designed to analyze the Ito sensitive to 4-AP in rat ventricular myocytes exposed to moderate concentrations of TNF-{alpha} (1–10 ng/ml) for 48 h. Figure 1A shows representative current traces obtained in a control cell incubated 48 h with vehicle (left) and in a cell exposed to TNF-{alpha} (5 ng/ml) (right). The amplitude of Ito was significantly reduced in cells treated with TNF-{alpha}. Figure 1B, left, illustrates the current density-voltage relationship obtained in 17 control (open circles) and 24 TNF-{alpha}-treated cells (solid circles). The voltage dependence was similar in both groups, but the current density was significantly smaller (P < 0.01, P < 0.001) from –20 to +60 mV in cells exposed to TNF-{alpha}.


Figure 1
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  		Fig. 1. Effect of tumor necrosis factor (TNF)-{alpha} exposure on transient outward K+ current (Ito) density and Kv4.2 protein expression in rat ventricular myocytes. A: family of Ito traces obtained in a control cell incubated 48 h with vehicle (left) [membrane capacitance (Cm) = 85 pF] and in a cell incubated 48 h with 5 ng/ml TNF-{alpha} (right) (Cm = 94 pF). Ito was evoked by depolarizing pulses from a holding potential of –80 mV to voltage tests from –30 to +60 mV in 10-mV increments for 500 ms; a prestep to –40 mV was used to inactivate sodium current. B, left: mean current density-voltage relation of Ito in 17 control cells ({circ}) and in 24 cells exposed to 5 ng/ml TNF-{alpha} (bullet). Right: concentration dependence of the TNF-{alpha}-induced decrease of Ito density at +30 mV. Cells were exposed to 4 different doses of TNF-{alpha}, 1, 2.5, 5, and 10 ng/ml during 48 h, and Ito was recorded at +30 mV in control and in TNF-{alpha}-treated ventricular myocytes. C, left: voltage dependence of Ito inactivation in 10 control ({circ}) and in 12 cells treated 48 h with 5 ng/ml TNF-{alpha} (bullet). I, current; Imax, maximum current. Inactivation of Ito was measured by a double protocol: a 2,000-ms conditioning prepulse from a holding potential of –80 mV to potentials between –100 and 0 mV (in 10-mV steps) was followed by a 500-ms depolarizing pulse to +40 mV. Right: Kv4.2 protein expression in control and in TNF-{alpha}-treated test samples. Top: representative immunoblot of Kv4.2 (~70 kDa) and its corresponding GAPDH (36 kDa). Bottom: histograms of the Kv4.2 protein expression (normalized to GAPDH) of 10 control (open bars) and 8 test samples exposed 48 h with 5 ng/ml TNF-{alpha} (solid bars). OD, optical density. Data are presented as percentage of values obtained in control and are expressed as means ± SE. *P < 0.05, **P < 0.01, ***P < 0.001.

 
Figure 1B, right, shows the concentration dependence of the effect of TNF-{alpha} exposure on Ito. Ventricular cells were exposed to vehicle (control) or to four different doses of TNF-{alpha}, 1, 2.5, 5, or 10 ng/ml for 48 h, and Ito was recorded. Ito density was measured at +30 mV in control cells (6.4 ± 1.2 pA/pF; n = 17) and in cells exposed to 1 ng/ml (4.9 ± 1.1 pA/pF; n = 13), 2.5 ng/ml (3.0 ± 0.7 pA/pF; n = 13), 5 ng/ml (2.2 ± 0.3 pA/pF; n = 24), and 10 ng/ml (1.8 ± 0.6 pA/pF; n = 10) TNF-{alpha}.

Figure 1C, left, illustrates the voltage dependence of Ito inactivation. Ventricular myocytes exposed to 5 ng/ml TNF-{alpha} showed a shift in the inactivation curve to more hyperpolarized potentials. Half-maximal inactivation was –49.9 ± 1.7 mV (n = 10) in control and –55.6 ± 1.6 mV (n = 12) in cells treated with TNF-{alpha} (P < 0.05), while the slope was similar in both groups: –4.8 ± 0.2 in control and –4.5 ± 0.4 in TNF-{alpha}-treated cells.

In mammalian hearts, the potassium channels responsible for the generation of Ito have been shown to be encoded by Kv1.4, Kv4.2, and Kv4.3 genes, although the relative contribution of each gene varies between species. In the rat ventricle, Kv4.2 has been postulated to be the primary determinant of Ito fast (or 4-AP-sensitive current) (11, 56). To determine whether TNF-{alpha} exposure could be associated with a downregulation of Ito channel expression, we analyzed the protein expression of the Kv4.2 channel in control vs. 5 ng/ml TNF-{alpha}-exposed cells. Figure 1C, top right, illustrates representative immunoblots from control and TNF-{alpha}-treated samples. Kv4.2 band was weaker in samples treated with TNF-{alpha} compared with control samples. Figure 1C, bottom right, shows that Kv4.2 protein expression level (normalized to GAPDH) was significantly lower (P < 0.05) in ventricular myocytes exposed for 48 h to TNF-{alpha}.

TNF-{alpha} increases APD. Our experiments show that 48-h exposure to TNF-{alpha} induced an important reduction of Ito density. To know whether this reduction could cause changes in the APD, we performed current-clamp experiments to analyze the characteristics of APs in seven control and in eight TNF-{alpha}-treated ventricular myocytes. Figure 2 illustrates representative superimposed traces of APs recorded in a control cell (open circle) vs. a cell exposed to TNF-{alpha} (solid circle). Cells treated with TNF-{alpha} showed prolonged APD compared with control. Exposure to TNF-{alpha} induced a significant increase of APD, measured at 20, 50, and 90% of repolarization (Fig. 2, right).


Figure 2
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  		Fig. 2. Effect of the TNF-{alpha} exposure on action potentials (APs). Left: superimposed APs obtained from a control cell incubated 48 h with vehicle ({circ}) and other cells exposed 48 h to 5 ng/ml TNF-{alpha} (bullet). Right: mean AP duration (APD) values measured at 20, 50, and 90% of repolarization (APD20, APD50, and APD90, respectively) in 7 control and in 8 TNF-{alpha}-exposed cells. Data are given as means ± SE. **P < 0.01, ***P < 0.001.

 
Effect of TNF-{alpha} on ICaL. To test whether TNF-{alpha} might induce changes in Ca2+ current (ICaL), we analyzed ICaL in 12 control and in 9 cells treated with 5 ng/ml TNF-{alpha}. Figure 3, top, shows similar ICaL recordings obtained in a control (left) and in a TNF-{alpha}-treated cell (right). Figure 3, bottom, shows similar ICaL density-voltage relationship obtained in control (open circles) and in TNF-{alpha}-treated cells (solid circles).


Figure 3
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  		Fig. 3. Effect of TNF-{alpha} exposure on L-type calcium current (ICaL) density in rat ventricular myocytes. Top: family of ICaL traces obtained in a control cell (left) (Cm = 96 pF) vs. a cell exposed 48 h to 5 ng/ml TNF-{alpha} (right) (Cm = 74 pF). ICaL was elicited by 200-ms steps from –40 mV to +60 mV. To inactivate the Na+ current, cells were predepolarized to –50 mV by a slow (500-ms) voltage ramp for 50 ms before each test depolarization was applied. Bottom: current density-voltage relation of ICaL in 12 control cells ({circ}) and in 9 cells treated 48 h with TNF-{alpha} 5 ng/ml (bullet). Data are given as means ± SE.

 
TNF-{alpha} increases the expression of hypertrophy gene markers. Several reports have proven that TNF-{alpha} is able to induce cardiac myocyte hypertrophy (41, 57). We next tested the potential involvement of TNF-{alpha} in this process. Reexpression of embryonic genes and transient activation of immediate early genes are frequently used indexes of myocyte hypertrophy (10, 39). Therefore, we analyzed the mRNA expression levels of an early gene, c-fos, and the marker of hypertrophy, ANF, in ventricular myocytes exposed to 5 ng/ml TNF-{alpha} for 48 h. Figure 4 shows that ANF mRNA (A) and c-fos mRNA levels (B) were significantly higher (P < 0.05) in myocytes treated with TNF-{alpha}.


Figure 4
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  		Fig. 4. TNF-{alpha} exposure induced the expression of hypertrophy gene markers c-fos and atrial natriuretic factor (ANF) in rat ventricular myocytes. Top: representative gel of ANF (A) and c-fos RT-PCR (B) obtained in a control sample incubated 48 h with vehicle and in a sample exposed 48 h to TNF-{alpha}. Ventricular cells isolated from aortic banded rats were used as a positive control (C+) (animals were killed 40 h after aortic stenosis). GAPDH was used as an internal standard. Bottom: histograms of ANF (A) and c-fos mRNA (B) expression of 5 control samples (open bars) and 5 samples incubated 48 h with TNF-{alpha} (solid bars). Data are presented as percentage of values obtained in control and are expressed as means ± SE. C– = negative control (no template was added). *P < 0.05.

 
Mechanisms involved in the decrease of Ito density induced by TNF-{alpha} exposure. Cytokines are known to enhance iNOS expression and oxidant species production in the heart. Figure 5 shows representative immunoblots demonstrating that iNOS expression was upregulated in ventricular myocytes exposed for 48 h to 5 ng/ml TNF-{alpha}. The next step was to determine whether iNOS and oxidant species formation were involved in the decrease of Ito density induced by TNF-{alpha} exposure. To test this hypothesis, recordings of Ito at +30 mV were obtained from control and TNF-{alpha}-treated ventricular myocytes preexposed to the selective iNOS inhibitor 1400-W (20 µM) (9). The results in Fig. 6 show that the decrease in Ito density induced by TNF-{alpha} exposure was fully reversed in ventricular myocytes treated with 1400-W or when cells were pretreated with the protein synthesis inhibitor CHX (20 µg/ml) (5). To investigate further whether oxidant species mediates TNF-{alpha}-induced Ito decrease, TNF-{alpha}-treated cells were preincubated with two different antioxidants, tocopherol (100 µM) (55) and the superoxide dismutase mimetic MnTBAP (100 µM) (21). Figure 6 shows that both antioxidants prevented TNF-{alpha}-induced Ito decrease.


Figure 5
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  		Fig. 5. TNF-{alpha} exposure induced inducible nitric oxide synthase (iNOS) protein expression in rat ventricular myocytes. Inset: representative immunoblot of iNOS (130 kDa) and its corresponding GAPDH (36 kDa). Lysates from rat lipopolysaccaride activated-macrophages (MCF) were used as a positive control. Figure shows the histograms of the mean protein levels of 5 control samples incubated 48 h with vehicle (open bars) and 5 samples incubated 48 h with TNF-{alpha} (solid bars). Data are presented as a percentage of values obtained in control and are expressed as means ± SE. **P < 0.01.

 

Figure 6
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  		Fig. 6. Involvement of the iNOS and oxidant species in the decrease of Ito induced by TNF-{alpha} exposure in rat ventricular myocytes. Ito density at +30 mV was measured in control cells, incubated 48 h with vehicle (n = 17), in ventricular myocytes exposed 48 h to TNF-{alpha} alone (n = 24) or in the presence of the selective iNOS inhibitor 1400-W (20 µM; n = 14), the inhibitor of protein synthesis cycloheximide (CHX; 20 µg/ml; n = 7), tocopherol (100 µM; n = 7), or the superoxide dismutase mimetic manganese(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP; 100 µM; n = 8). Values of Ito density obtained from control cells treated with inhibitors and antioxidants were not significantly different from values of Ito obtained in control cells without treatment. The bar graphs show the percentage of change with respect to each control group. Data are given as means ± SE. ***P < 0.001 vs. TNF-{alpha} alone.

 
The results obtained support the idea that reactive nitrogen and oxygen species are involved in the mechanism of TNF-{alpha}-induced Ito decrease. The combination of NO and superoxide anion forms the highly reactive oxidant peroxynitrite, a powerful oxidant that has been proven to impair the function of multiple proteins, including vascular potassium channels (32). To test whether peroxynitrite can modulate Ito in rat ventricular myocytes, we analyzed the values of Ito density in cells exposed to 300 µM peroxynitrite.

Figure 7 illustrates the results obtained in this group of experiments. Panel A shows a family of representative Ito traces obtained in a control cell (left) and in a cell exposed to peroxynitrite (right). Panel B shows the density-voltage relationship obtained in 13 control vs. 10 cells exposed to peroxynitrite. Ito density was significantly smaller (from –20 to +60 mV) in cells exposed to peroxynitrite, in agreement with the possible participation of peroxynitrite in the decrease of Ito induced by TNF-{alpha} observed in our study.


Figure 7
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  		Fig. 7. Effect of peroxynitrite on Ito. A: sample traces of Ito currents obtained in a control cell (left) (Cm = 130 pF) and in a cell exposed for 3–15 min to peroxynitrite (right) (Cm = 95 pF). Ito was evoked using the protocol that appears in the inset. B: mean density-voltage relation of Ito in 13 control cells ({circ}) and in 10 cells exposed to 300 µM peroxynitrite (bullet). The peroxynitrite concentration in the stock solution was determined spectrophotometrically before each experiment. *P < 0.05, **P < 0.01.

 

   DISCUSSION
TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
In the present study, we have explored the effect of 48-h exposure of TNF-{alpha} on Ito in rat ventricular myocytes.

Our results demonstrate that TNF-{alpha} is able to trigger hypertrophic-related responses, producing a significant reduction of Ito density and modifying its inactivation. Also, TNF-{alpha} prolongs the APD and downregulates Kv4.2 protein expression. The present study also shows that iNOS expression and oxidant species generation participate in the electrophysiological effects induced by this cytokine and reports for the first time that acute peroxynitrite exposure can impair Ito in cardiac myocytes.

Deleterious cardiac effects of proinflammatory cytokines have been associated with high concentrations or long exposure to TNF-{alpha} (36, 42). Cardiac hypertrophy, which appears initially as a compensatory mechanism, is a common feature of many cardiovascular diseases, including heart failure (15). Relevant concentrations of TNF-{alpha} can induce hypertrophic response in cardiac myocytes (41, 57) and in transgenic mice overexpressing TNF-{alpha} in the myocardium, consistently demonstrating myocardial hypertrophy (8, 29, 52). The present study also confirms that moderate concentrations of TNF-{alpha} evoke the hypertrophic response in ventricular myocytes. ANF and protooncogenes such as c-fos have been identified as markers of hypertrophic gene programs (51), and, in our study, both genes were upregulated in the ventricular myocytes exposed to TNF-{alpha}.

Compensated cardiac hypertrophy is related to a decrease in potassium currents and prolonged APD (4, 6, 23, 31). Ventricular myocytes exposed to TNF-{alpha} showed similar alterations. Ito density was found to be diminished, and this effect was concentration dependent (from 1 to 10 ng/ml). Although limitations related to the use of room temperature and whole cell patch-clamp technique have to be taken into account, our results also demonstrated that APDs were prolonged in TNF-{alpha}-treated myocytes. Furthermore, the decrease of Ito induced by TNF-{alpha} was the consequence of two mechanisms: a shift in the inactivation curve to more hyperpolarizing potentials and a significant reduction of Kv4.2 channel expression.

Another possible mechanism involved in the effect of TNF-{alpha} on APD prolongation could be that this cytokine induces changes in ICaL. Therefore, the present study has also analyzed ICaL in ventricular myocytes exposed to TNF-{alpha}. Our results show that the density and voltage dependence of this current were not modified by TNF-{alpha} exposure. Similar results have been obtained with moderate concentrations of TNF-{alpha} (10–25 ng/ml) (53, 57), while a decrease of ICaL has been reported in the presence of high concentrations of TNF-{alpha} (>300 ng/ml) (28).

Several reviews have been published dealing with the role of proinflammatory cytokines in cardiovascular diseases (36, 38, 47). However, a possible mechanism involving the direct effects of moderate concentrations of cytokines on cardiac K+ channels has barely been explored (55). So far, only two studies have been published analyzing the direct effect of TNF-{alpha} on cardiac potassium channels. Wang et al. (55) demonstrated a reduction of human ether-a-go-go-related gene and rapid delayed-rectifier K+ currents using 50–100 ng/ml TNF-{alpha}. More recently, Kawada et al. (25) showed a reduction of Ito in neonatal cells incubated with 50 ng/ml of TNF-{alpha}, although the mechanisms implicated were not explored.

One of the mechanisms associated with the effects of cytokines on cardiac diseases is their capacity for iNOS induction and generation of oxidant species. In the isolated, working rat heart, cytokine perfusion stimulates the myocardial expression of iNOS and the production of superoxide enhancing peroxynitrite generation (13). The evidence obtained in our study showing that iNOS expression was upregulated by TNF-{alpha} made us hypothesize that iNOS-derived oxidant species could play a role in the downregulation of Ito induced by TNF-{alpha}. Interestingly, when iNOS activity or its induced expression was suppressed by the selective iNOS inhibitor 1400-W or the inhibitor of protein synthesis CHX, respectively, the effect of TNF-{alpha} on Ito was prevented. Upregulation of iNOS expression by cytokines produces large amounts of NO and reduces molecular oxygen to superoxide (especially in conditions of substrate or cofactor deficiency) (2).

Oxidant stress is implicated in cardiovascular diseases, including hypertension, atherosclerosis, and ischemia-reperfusion (24, 44). Oxidant species production takes part in the regulation of several gene expression and cell growth (30, 48). Additionally, oxidant species are involved in the regulation of hypertrophic and apoptotic signaling pathways in cardiac myocytes (50). In this sense, evidence exists that antioxidants can inhibit TNF-{alpha}-induced hypertrophy in cultured rat cardiac myocytes (41). Furthermore, several studies have addressed the fact that oxidant species are able to induce changes in the function and expression of several ion channels (20, 22, 27, 34). The results obtained in our study using the antioxidants {alpha}-tocopherol and the superoxide dismutase mimetic MnTBAP strongly suggest that oxidant species are involved in the effect of TNF-{alpha} on Ito. The fact that iNOS inhibition as well as antioxidant maneuvers reverse the effect of TNF-{alpha} on Ito suggests the involvement of peroxynitrite. This compound is a highly reactive oxidant, resulting from the reaction between NO and superoxide (3), which shows important biological properties relevant to the cardiovascular system (49). Indeed, we have also shown that authentic peroxynitrite directly inhibits Ito, supporting the idea that this oxidant might participate in the downregulation of Ito induced by TNF-{alpha} exposure in cardiac ventricular myocytes. Moreover, the fact that peroxynitrite's effect on Ito was observed in a very short time precludes transcriptional regulation of Kv4.2 channels and suggests some kind of posttranslational modification of the channel, i.e., nitrosylation, nitration, and oxidation of key amino acid residues (37). However, reactive oxygen and nitrogen species production may alter the activity of other signaling mechanisms that secondarily lead to changes in Kv4.2 channel activity or channel gene expression. It has been described recently that redox mechanisms can be involved in controlling gene expression of Kv1.4 and Kv4.2 channels in ventricular myocytes (34).

In summary, we conclude that TNF-{alpha} exposure promotes electrophysiological changes (decreased Ito and APD prolongation) in rat ventricular myocytes, through iNOS induction and generation of oxidant species, providing new insights into how cytokines can modulate K+ channels in the heart.


   GRANTS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This study was supported by the Ministerio de Educación of Spain (BFI2002-00536 and SAF-2005-01887). M. Fernández-Velasco was a fellow of Consejo Superior de Investigaciones Científicas. G. Ruiz-Hurtado is a graduate research fellow of the Ministerio de Educación of Spain.


   ACKNOWLEDGMENTS
 
We are grateful to M. Bas, F. Ortego, and M. L. Hidalgo for excellent technical assistance. We also thank Dr. A. M. Gómez and Dr. A. Rueda for careful reading of this manuscript.


   FOOTNOTES
 

Address for reprint requests and other correspondence: C. Delgado, Institute of Pharmacology and Toxicology (CSIC-UCM), School of Medicine, Universidad Complutense, 28040 Madrid, Spain (e-mail: cdelgado{at}med.ucm.es)

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.

* M. Fernández-Velasco and G. Ruiz-Hurtado contributed equally to this work. Back


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