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Cardiovascular Research Advance Access first published online on August 4, 2008
This version [Corrected Proof] published online on August 24, 2008
Cardiovascular Research, doi:10.1093/cvr/cvn205
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org

Nitric oxide inhibits Kv4.3 and human cardiac transient outward potassium current (Ito1)

Ricardo Gómez1,{dagger}, Lucía Núñez1,{dagger}, Miguel Vaquero1, Irene Amorós1, Adriana Barana1, Teresa de Prada2, Carlos Macaya2, Luis Maroto3, Enrique Rodríguez3, Ricardo Caballero1,*, Antonio López-Farré2, Juan Tamargo1 and Eva Delpón1

1 Department of Pharmacology, School of Medicine, Universidad Complutense, 28040 Madrid, Spain
2 Cardiovascular Research Unit, Hospital Clínico San Carlos, Madrid, Spain
3 Cardiac Surgery Department, Hospital Clínico San Carlos, Madrid, Spain

* Corresponding author. Tel: +34 34913941474; fax: +34 913941470. E-mail address: rcaballero{at}med.ucm.es

Received 3 March 2008; revised 18 July 2008; accepted 24 July 2008

Time for primary review: 27 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: Chronic atrial fibrillation (CAF) is characterized by a shortening of the plateau phase of the action potentials (AP) and a decrease in the bioavailability of nitric oxide (NO). In this study, we analysed the effects of NO on Kv4.3 (IKv4.3) and on human transient outward K+ (Ito1) currents as well as the signalling pathways responsible for them. We also analysed the expression of NO synthase 3 (NOS3) in patients with CAF.

Methods and results: IKv4.3 and Ito1 currents were recorded in Chinese hamster ovary cells and in human atrial and mouse ventricular dissociated myocytes using the whole-cell patch clamp. The expression of NOS3 was analysed by western blotting. AP were recorded using conventional microelectrode techniques in mouse atrial preparations. NO and NO donors inhibited IKv4.3 and human Ito1 in a concentration- and voltage-dependent manner (IC50 for NO: 375.0 ± 48 nM) as a consequence of the activation of adenylate cyclase and the subsequent activation of the cAMP-dependent protein kinase and the serine–threonine phosphatase 2A. The density of the Ito1 recorded in ventricular myocytes from wild-type (WT) and NOS3-deficient mice (NOS3–/–) was not significantly different. Furthermore, the duration of atrial AP repolarization in WT and NOS3–/– mice was not different. The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atrial AP and lengthened the AP duration measured at 20 and 50% of repolarization of the human atrial CAF-remodelled AP as determined using a mathematical model. However, the expression of NOS3 was not modified in left atrial appendages from CAF patients.

Conclusion: Our results suggested that the increase in the atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the Ito1 as a result of the activation of non-canonical enzymatic pathways.

KEYWORDS Atrial fibrillation; Nitric oxide; Human transient outward current; Patch clamp; Kv4.3 channels


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Atrial fibrillation (AF), the most common sustained cardiac arrhythmia, alters atrial electrophysiology in a way that promotes its occurrence and persistence, a phenomenon called ‘electrical remodelling’, which is mainly characterized by a marked shortening of the plateau and the final phase of repolarization of human atrial action potential (AP).1

Nitric oxide (NO), a diffusible messenger produced by all myocardial cell types, has important effects on cardiac function including rate, contraction, relaxation, coronary reserve, platelet aggregation, mitochondrial respiration, cell growth, and survival.2 In a porcine model, AF induced an ‘endocardial dysfunction’, characterized by a marked decrease in NO synthase 3 (NOS3) expression and the corresponding drop in NO concentration.3 Currently, it is unknown whether human chronic AF (CAF) also decreases NOS3 atrial expression, but it is clearly established, both in patients and in animal models, that NO bioavailability decreases in AF because of an increased atrial oxidative stress.4,5

The height and the duration of the plateau phase of the human atrial AP are determined by the balance between the inward L-type Ca2+ current (ICaL) and several outward K+ currents.6 The effects of NO on ICaL have been studied in cardiac myocytes from different species and in cloned channels expressed in heterologous systems.7,8 Previous reports demonstrated that NO inhibits the rapid component of the delayed rectifier current (IKr),9 whereas it increases the slow component (IKs).10 More recently, we have described that NO blocks hKv1.5 channels, which generate the ultrarapid delayed rectifier K+ current (IKur).11 However, the effects of NO on the Ca2+-independent component of the transient outward K+ current (Ito1) are presently unknown. Human cardiac Ito1 is predominantly carried by Kv4.3 {alpha}-subunits, which assemble with KChIPs, DPP6, and probably also MiRPs ancillary subunits.12 Thus, this study aimed to analyse the effects of NO on Kv4.3 current (IKv4.3) and on human atrial Ito1, as well as the intracellular signalling pathway responsible for the effects. Our results suggested that the increase in atrial NO bioavailability could partially restore the duration of the plateau phase of CAF-remodelled AP by inhibiting the Ito1 as a result of the activation of non-canonical enzymatic pathways.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
The study was approved by the Ethics Committee of the Hospital Clínico San Carlos de Madrid (No. B-05/017) and conforms with the principles outlined in the Declaration of Helsinki. Each patient gave written informed consent.

2.1 Western blotting of human atrial appendages
NOS3 expression was determined in left atrial appendages (LAA) from sinus rhythm (SR) or CAF patients by western blot analysis as described.3 Patient characteristics are shown in Table 1. To determine S-nitrosylated Kv4.3 channels, the biotin-switch assay followed by western blot was performed.11


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  		Table 1 Patient characteristics

 
2.2 Cell culture, human and mouse myocyte isolation, and solutions
Chinese hamster ovary (CHO) cells were transiently transfected with the cDNA encoding Kv4.3 as described previously.13,14 Cells were perfused with an external solution containing (mM): NaCl 136, KCl 4, CaCl2 1.8, MgCl2 1, HEPES 10, and glucose 10 (pH 7.4 with NaOH). Human atrial myocytes were isolated from LAA obtained from patients in SR undergoing cardiac surgery as described.15 For each group of experiments, myocytes were obtained from at least four patients. Mouse ventricular myocytes were enzymatically isolated11,13,14 from wild-type (WT) (C57BL/6J strain) and NOS3-deficient (NOS3–/–) 12-week-old mice (The Jackson Laboratory). The investigation conforms with the Guide for the Care and Use of Laboratory Animals (NIH Publication No. 85-23, revised 1996). For measurements of human and mouse Ito1, 2 mM CoCl2 was added to the external solution for blocking ICaL. Recording pipettes were filled with internal solution containing (mM): K-aspartate 80, KCl 42, KH2PO4 10, MgATP 5, phosphocreatine 3, HEPES 5, and EGTA 5 (pH 7.2 with KOH). The NO donors (±)-S-nitroso-N-acetylpenicillamine (SNAP, Calbiochem) and 2-(N, N-diethylamine)-diazenolate-2-oxide (DEANO, Sigma) were initially dissolved in ethanol and water, respectively, to yield 10 mM stock solutions. NO donor solutions were freshly prepared for each experiment, and the NO-saturated solution was prepared as described previously.11 NO concentration in the perfusing chamber was measured using a potentiometric NO sensor.

2.3 Recording techniques
IKv4.3 and Ito1 were recorded using the whole-cell patch clamp technique at 21–23°C with Axopatch-200B amplifiers (Molecular Devices). Capacitance and series resistance were optimized and ~80% compensation was usually obtained. In CHO cells, mean maximum IKv4.3 amplitude, uncompensated access resistance (Ra), and cell capacitance (Cm) were 2.5 ± 0.4 nA, 1.7 ± 0.2 M{Omega}, and 14.8 ± 0.7 pF, respectively (n = 37). In human atrial myocytes, the maximum outward K+ current amplitude averaged 798.5 ± 126.9 pA, whereas the mean values of Cm and Ra were 115.3 ± 16.2 pF and 2.0 ± 0.3 M{Omega}, respectively (n = 21). There were no differences in the Cm from WT and NOS3–/– myocytes (126 ± 17 pF). In these cells, maximum outward current and Ra were 2.3 ± 0.3 nA and 1.6 ± 0.1 M{Omega}, respectively (n = 12). Thus, under our experimental conditions, no significant voltage errors (<5 mV) due to series resistance were expected with the micropipettes used (tip resistance < 3.5 M{Omega}).

AP were recorded at 34°C in mouse left atria superfused with Tyrode solution (mM: NaCl 137, KCl 5.4, MgCl2 1.0, CaCl2 1.8, NaH2PO4 0.42, NaHCO3 11, and glucose 10), bubbled with 95% O2/5% CO2, and driven at 3 Hz using conventional microelectrode techniques.14

2.4 Mathematical modelling of a human atrial action potential
For simulating the shapes of human atrial AP under non-AF- and AF-modified conditions, we employed a mathematical model. To simulate the electrophysiological remodelling in CAF, reductions of 70% in ICaL, and of 50% in Ito1 and IKur, were introduced as described.16 The model was run at a frequency of 1 Hz in the absence and presence of 200 nM NO by incorporating the specific conductance and time- and voltage-dependent modifications induced by NO on Ito1, IKur, IKr, IKs, ICaL, and INa.

2.5 Statistical methods
Results are expressed as mean ± SEM. Data were compared by ANOVA followed by the Newman–Keuls test. A value of P < 0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Effects of nitric oxide and nitric oxide donors on IKv4.3
Figure 1A shows IKv4.3 traces recorded by applying 250 ms pulses from –80 to +50 mV under control conditions and in the presence of 200 µM SNAP. The NO donor reduced the peak IKv4.3 by 30.8 ± 4.5% (n = 7) without modifying the time course of inactivation measured from the bi-exponential fit to the current traces (Table 2). SNAP reduced the K+ efflux crossing the membrane through Kv4.3 channels (charge), estimated from current–time integrals at +50 mV, by 37.5 ± 1.3%, a reduction not significantly different from that measured at the peak (P > 0.05). For this reason, the reduction of the peak current amplitude was used as an index of steady-state inhibition. Figure 1B shows the time course of NO release during the development of the experiment (Figure 1A). NO concentration reached a maximum within 3–4 min (192 ± 15 nM, n = 7) and remained stable during SNAP perfusion (8–10 min), decreasing to basal levels after perfusion with SNAP-free solution.


Figure 1
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  		Figure 1 IKv4.3 recorded in Chinese hamster ovary cells in the absence, presence, and after washout of S-nitroso-N-acetylpenicillamine (SNAP) (A), 2-(N, N-diethylamine)-diazenolate-2-oxide (DEANO) (C), and nitric oxide (NO)-saturated solution (D). (B) Nitric oxide concentrations yielded before and after the perfusion of S-nitroso-N-acetylpenicillamine measured using a potentiometric nitric oxide sensor. Percentage of IKv4.3 reduction induced by S-nitroso-N-acetylpenicillamine as a function of S-nitroso-N-acetylpenicillamine concentrations (E) and the released nitric oxide (F). Continuous lines represent the Hill equation fit to the data. Each point represents the mean ± SEM of five or more experiments.

 


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  		Table 2 Effects of nitric oxide and nitric oxide donors on IKv4.3

 
The effects of another NO donor, DEANO (3 µM), and an NO-saturated solution were also studied. In these experiments, the concentration of NO released averaged 274 ± 18 (n = 6) and 219 ± 10 nM (n = 6), respectively. DEANO (Figure 1C) and the NO solution (Figure 1D) reduced the peak IKv4.3 by 35.5 ± 8.1 and 28.0 ± 5.8%, respectively. These values were not different from those obtained with the concentration of SNAP that released the same amount of NO (P > 0.05), indicating that, in all cases, the inhibition of IKv4.3 was exclusively due to NO. Moreover, neither DEANO nor the NO solution modified the time course of current decay (Table 2).

The Hill equation was used to fit the concentration dependence of peak current inhibition at +50 mV, yielding an IC50 of 391.8 ± 49.6 µM for SNAP (nH = 1.3 ± 0.2) (Figure 1E). When the inhibition was plotted as a function of the released NO by SNAP (Figure 1F), the IC50 was 375.0 ± 47.8 nM (nH = 1.2 ± 0.2).

Figure 2A shows IKv4.3 traces obtained by applying 250 ms pulses from –80 mV to potentials between –90 and +50 mV (conditioning pulse) followed by a test pulse to +50 mV in the absence and presence of 200 µM SNAP. Current–voltage relationships (Figure 2B) show that SNAP significantly decreased peak IKv4.3 at potentials positive to –20 mV (P < 0.05). This reduction appeared in the range of potentials coinciding with that of channel activation (inhibition at –10 mV: 30.2 ± 5.5%), remaining unchanged at more positive potentials (P > 0.05). Inactivation curves (Figure 2C) were constructed by plotting the peak current amplitude elicited by the test pulse as a function of the conditioning pulse and by fitting a Boltzmann function to the data. SNAP decreased IKv4.3 between –90 and –30 mV and shifted Vh towards negative potentials without modifying the slope of the curve (Table 2). These voltage-dependent effects were also corroborated when DEANO and the NO solution were perfused (Table 2). Relative current was plotted as a function of the conditioning pulse, demonstrating that the inhibition remained unchanged at potentials between –90 mV (30.1 ± 4.5%) and –60 mV (34.0 ± 5.0%, P > 0.05) and, thereafter, it progressively increased at more positive potentials reaching a 62.3 ± 4.8% at –30 mV (P < 0.05 vs. inhibition at –90 mV).


Figure 2
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  		Figure 2 (A) IKv4.3 recorded in the absence and presence of S-nitroso-N-acetylpenicillamine (SNAP). (B) Mean IKv4.3 current–voltage relationships in the absence and presence of S-nitroso-N-acetylpenicillamine. (C) Inactivation curves of Kv4.3 channels in the absence and presence of S-nitroso-N-acetylpenicillamine. A Boltzmann equation was fitted to the data (continuous lines). The dashed line represents the curve in the presence of drug, normalized to the control amplitude. In (B) and (C), squares represent the relative current as a function of the membrane potential. Each point represents the mean ± SEM of five or more experiments. *P < 0.05 vs. control. **P < 0.01 vs. control. #P < 0.05 vs. inhibition at –90 mV.

 
3.2 Signalling pathways involved in the inhibition of IKv4.3
The guanylate cyclase (GC)/cGMP/PKG pathway is the most common mechanism implicated in NO effects.2 To test the involvement of this pathway, the effects of SNAP in the presence of the GC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) were studied. Figure 3A shows that 50 µM ODQ decreased the peak IKv4.3 at +50 mV by 27.0 ± 6.7%. The addition of SNAP (200 µM) further decreased the current by 29.2 ± 7.2% (n = 5), which was the same inhibition as that produced by SNAP alone (P > 0.05, Figure 3C). In the same way, after the perfusion with 500 µM 8-bromo-cGMP, a potent cell-permeable PKG activator, SNAP produced a 26.7 ± 4.9% inhibition of the current (n = 4, P < 0.05). These results suggested that the GC/cGMP/PKG pathway was not involved. To confirm that these results were not due to the expression system, we studied the effects of SNAP on Kv4.3-carried Ito1 recorded in mouse ventricular myocytes by applying 250 ms pulses from –80 to +50 mV in the presence of 50 µM 4-aminopyridine (4-AP), which at this concentration, selectively inhibits IKur.6 Under these conditions, 200 µM SNAP decreased Ito1 by 27.3 ± 3.5% (not shown), and when SNAP was superfused in the presence of ODQ (inset in Figure 3A), the inhibition (30.9 ± 4.4%, n = 5) was not different from that produced by SNAP alone (P > 0.05). These data confirmed that the GC/cGMP/PKG pathway was not involved in the effects of NO on IKv4.3.


Figure 3
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  		Figure 3 (A) IKv4.3 recorded in a Chinese hamster ovary cell in the absence and presence of ODQ alone and plus S-nitroso-N-acetylpenicillamine (SNAP). The inset shows Ito1 traces recorded in a mouse ventricular myocyte in the presence of 4-AP + ODQ alone and plus S-nitroso-N-acetylpenicillamine. (B) Western blots showing S-nitrosylated Kv4.3, Kv4.3, and β-actin expression, in control conditions or after treatment with 200 µM S-nitroso-N-acetylpenicillamine and 3 µM 2-(N, N-diethylamine)-diazenolate-2-oxide (DEANO). (C) Percentage of inhibition produced either by S-nitroso-N-acetylpenicillamine alone or by S-nitroso-N-acetylpenicillamine in the presence of different inhibitors of enzymatic pathways. IKv4.3 recorded in control conditions and in the presence of (D) CPA or (E) PKI alone and plus S-nitroso-N-acetylpenicillamine. (F) Signalling pathway responsible for the nitric oxide effects. (G) IKv4.3 recorded in control conditions and in the presence of okadaic acid (OKA) + PKI without or with S-nitroso-N-acetylpenicillamine. *P < 0.05 and **P < 0.01 vs. S-nitroso- N-acetylpenicillamine alone.

 
S-Nitrosylation, which is highly dependent on the cellular redox state, modifies the function of many proteins, including several receptors (ryanodine and neuronal NMDA)17 and channels (hKv1.5).11 Therefore, the effects of SNAP in the presence of the thiol-specific reducing agent, dithiothreitol (DTT), were studied. DTT reduced IKv4.3 by 35.4 ± 6.4% and the addition of SNAP further decreased the current by 25.8 ± 6.1% (Figure 3C, n = 5, P > 0.05), an inhibition similar to that produced in the absence of DTT. Therefore, S-nitrosylation was not involved in the NO effects on IKv4.3. However, the analysis of the amino acid sequence of Kv4.3 protein revealed at least two Cys (C131 and C132) located in an acid–base micro-environment, which can act as a catalyst for nitrosylation–denitrosylation of thiol residues.17 A biotin switch assay followed by a western blot confirmed the presence of S-nitrosylated Cys residues on the Kv4.3 protein in donor-treated and untreated cells (Figure 3B). These results suggest that Kv4.3 channels belong to the ‘nitrosylome’, the pool of proteins that are S-nitrosylated in cells.17

The involvement of either the PKC or the Ca2+/calmodulin-dependent protein kinase II (CaMKII) activation in the SNAP-induced inhibition was also discarded by performing experiments in the presence of the PKC inhibitor staurosporine (1 µM) or KN-93 (10 µM), a specific CaMKII inhibitor, respectively (Figure 3C).

To study whether the adenylate cyclase (AC)/cAMP/PKA pathway was involved in the inhibition of IKv4.3, we analysed the effects of SNAP in the presence of a membrane permeable (9-cyclopentyladenine, CPA, 100 µM) or an impermeable (2,5-dideoxy-ATP, ddATP, 200 nM) AC inhibitor, the latter being added to the internal solution. CPA (Figure 3D) and ddATP decreased IKv4.3 by 27.2 ± 4.8 (n = 4) and 20.4 ± 4.3% (n = 4), respectively, and the addition of 200 µM SNAP produced no effect on IKv4.3 (1.2 ± 2.5 and 1.9 ± 2.3%, respectively, Figure 3C). These results demonstrated that the SNAP-induced effects were mediated by the activation of the AC. In fact, after perfusion with 8-bromo-cAMP (500 µM), a cell-permeable cAMP analogue that inhibited IKv4.3 by 30.3 ± 2.3% (n = 4), SNAP did not further modify the IKv4.3 amplitude (2.8 ± 0.7%). However, in the presence of 1 µM PKI (a PKA inhibitor), SNAP decreased IKv4.3 by 16.9 ± 3.2% (Figure 3E), an inhibition significantly smaller than that produced by SNAP alone (Figure 3C, n = 6, P < 0.05). So, the effects of NO on Kv4.3 channels were only partially mediated via AC/cAMP/PKA.

It has been described that a cAMP-dependent methylation can increase the catalytic activity of serine/threonine phosphatase type 2A (PP2A),18 and that the activation of PP2A inhibits Ito1.13 Therefore, we also studied the effects of SNAP in the presence of okadaic acid (OKA), a PP2A inhibitor. OKA (10 nM) inhibited IKv4.3 by 15.4 ± 4.8%, and the addition of SNAP further decreased the current by 18.1 ± 4.6%, an inhibition significantly lower than that produced by SNAP alone (Figure 3C, n = 5, P < 0.05). Similar results were obtained when cantharidin was used as a PP2A inhibitor (17.8 ± 6.1%, n = 6, Figure 3C). Finally, to discard the putative implication of the serine/threonine phosphatase type 2B (calcineurin), which is known to be modulated by NO,19 a group of experiments were performed in the presence of cyclosporin (10 µM), which inhibits calcineurin. SNAP inhibited IKv4.3 in a similar way, both in the presence and absence of cyclosporin, which inhibited the current by 8.8 ± 1.9% (n = 4) (Figure 3C), discarding the implication of calcineurin on the NO inhibitory effects.

All these results demonstrated that the effects produced by SNAP were mediated by the activation of AC, which simultaneously activates the PKA and the PP2A (Figure 3F). This was further demonstrated because the simultaneous presence of PKI and OKA completely prevented the SNAP-induced inhibition (Figures 3C and G).

3.3 Effects of nitric oxide on Ito1 on human atrial myocytes
We next studied the effects of SNAP on Ito1 recorded in dissociated human LAA myocytes. A 25 ms prepulse to –30 mV was applied to inactivate INa, followed by a 250 ms pulse to +50 mV, eliciting an outward K+ current which is composed of at least two currents: Ito1 and IKur. In the presence of 50 µM 4-AP (to block IKur), SNAP 200 µM decreased Ito1 amplitude by 24.9 ± 1.2% without modifying the time course of current decay ({tau}CON = 39.9 ± 2.9 ms vs. {tau}SNAP = 44.5 ± 5.8 ms, P > 0.05, n = 5) (Figures 4A and F).


Figure 4
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  		Figure 4 Effects of S-nitroso-N-acetylpenicillamine (SNAP) on Ito1 recorded in human atrial myocytes in the presence of 4-AP (A), 4-AP + CPA (B), 4-AP + ddATP (C), 4-AP + PKI (D), or 4-AP + okadaic acid (OKA) (E). (F) Percentage of human Ito1 inhibition produced by S-nitroso-N-acetylpenicillamine alone or in the presence of the different inhibitors of enzymatic pathways. (G) Nitric oxide synthase 3 and β-actin expression in left atrial appendages from sinus rhythm (SR) and chronic atrial fibrillation (CAF) patients and densitometric measurement of nitric oxide synthase 3 expression in left atrial appendages from sinus rhythm (n = 5) and chronic atrial fibrillation (n = 7) patients. *P < 0.05 and **P < 0.01 vs. S-nitroso-N-acetylpenicillamine alone.

 
Figures 4B, C, and F show that the inhibition of AC, with 100 µM CPA or 200 nM ddATP, also completely abolished the effects of SNAP on human Ito1. To test whether PKA activation was responsible for part of the effects, we analysed the effects of SNAP in the presence of 1 µM PKI (Figure 4D). Under these conditions, SNAP decreased the current by 11.5 ± 2.9% (n = 4), contrasting with the 24.9 ± 1.2% produced by SNAP alone (P < 0.05) (Figure 4F). Moreover, superfusion of OKA partially prevented the inhibition produced by SNAP (13.2 ± 4.9%, n = 4, Figure 4F), demonstrating that the activation of PP2A was also implicated (Figure 4E).

3.4 Determination of the expression of nitric oxide synthase 3 in human atria
We also tested whether CAF could modify NOS3 expression. Figure 4G shows representative NOS3 western blots from LAA obtained from CAF and SR patients. The densitometric analysis revealed that the expression was not statistically different between the two groups (P > 0.05, n = 11).

3.5 Ito1 and action potential characteristics in nitric oxide synthase 3-deficient mice
To elucidate whether the decrease in myocardial NO concentrations affected the Ito1, we compared the Ito1 recorded in isolated ventricular myocytes from CD57 WT and NOS3–/– mice. Figure 5A shows families of currents recorded in the presence of 4-AP. The Ito1 density–voltage relationships demonstrated that there were no differences in current density (Ito1 amplitude/Cm) between the two groups (Figure 5B). Similarly, inactivation time course ({tau}WT = 39.4 ± 5.2 ms and {tau}NOS3–/– = 41.1 ± 3.6 ms) and the voltage dependence of Ito1 inactivation (VhWT = –33.8 ± 2.4 and VhNOS3–/– = –38.9 ± 2.2 mV, Figure 5C) were not different (n = 6 in each group, P > 0.05).


Figure 5
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  		Figure 5 (A) Representative Ito1 traces recorded in wild-type (WT) (left) and nitric oxide synthase 3-deficient (NOS3–/–) (right) mouse ventricular myocytes in the presence of 4-AP. (B) Mean Ito1 density-voltage relationships in wild-type and nitric oxide synthase 3-deficient mouse myocytes. (C) Inactivation curves of Ito1 in wild-type and nitric oxide synthase 3-deficient mouse myocytes. A Boltzmann equation was fitted to the data (continuous lines). (D and E) Action potentials recorded in left atrial multicellular preparations from wild-type (D) and nitric oxide synthase 3-deficient (E) mice in the absence and presence of S-nitroso-N-acetylpenicillamine (SNAP). (F) APD measured at 20, 50, and 90% of repolarization in left atria from wild-type and nitric oxide synthase 3-deficient mice. (G) Percentage of change in the APD produced by S-nitroso-N-acetylpenicillamine in wild-type and nitric oxide synthase 3-deficient mice. Each point and bar represents the mean ± SEM of six or more experiments.

 
WT and NOS3–/– multicellular atrial preparations were used for recording AP (Figure 5D and E). There was no difference between WT and NOS3–/– atrial preparations in the AP duration measured at 20 (APD20), 50 (APD50), and 90% (APD90) of repolarization (Figure 5F). In contrast, AP amplitude (APA) was significantly lower in NOS3–/– (104.6 ± 3.9 mV, n = 6) than in WT preparations (119.2 ± 3.1 mV, n = 6, P < 0.05), even when no differences in the resting membrane potential (RMP) were found (RMP NOS3–/– = –83.2 ± 3.6 vs. RMP WT = –86.7 ± 3.1 mV, n = 6, P > 0.05). Finally, the effects of 200 µM SNAP on the AP characteristics were tested (Figure 5D and E). Both groups of atrial preparations responded identically to the increase in NO concentration. SNAP significantly decreased the APA by 6.0 ± 1.6 mV (P < 0.05) and hyperpolarized the RMP by 3.5 ± 1.2 mV (P < 0.05). Repolarization was also affected by SNAP, so that APD20 was significantly prolonged by ~25% (from 6.3 ± 0.8 to 7.8 ± 1.0 ms in WT and from 6.0 ± 0.7 to 7.5 ± 0.9 ms in NOS3–/– mice, respectively, P < 0.05), whereas APD50 and APD90 were significantly shortened (Figure 5G).

3.6 Effects of nitric oxide on a mathematical model of chronic atrial fibrillation-modified action potentials
We ran a previously described model of a human atrial AP in SR and in CAF-simulated conditions.16 To simulate the electrophysiological remodelling in CAF, reductions of 70% in ICaL, and of 50% in Ito1 and IKur, were introduced as described (Figure 6C and D). These modifications produced indirect changes of other currents (i.e. a reduction in IKr, IKs, and INaCa) (compare panels E and F), resulting in the characteristic triangular shape and shortening of APs in CAF.16 Figure 6 shows the AP in SR (panel A) and CAF conditions (panel B) in the absence of (continuous line), and after increasing, the NO concentration to 200 nM (dashed line). The latter was run by incorporating the effects produced by NO ~200 nM in the currents shown in Figures 6C–F. Ito1 was decreased by 30.8% as described in this paper. For the other currents, we introduced the data previously reported in the literature regarding the effects of NO. Indeed, 200 µM SNAP decreased ICaL, IKur, and IKr by 40, 31, and 45%, respectively.8,9,11 Moreover, 200 nM NO reduced INa by 5%,20 whereas 200 µM sodium nitroprusside increased IKs by 20%.10 In non-AF conditions, NO-combined blockade of Ito1, IKur, ICaL, and IKr produced a dynamic change in IKs which overcame the NO-induced increase in this latter current. The global result was a slight prolongation of the APD20 and a shortening of the APD50 by 12.5%. In CAF conditions, NO blockade of IKur and Ito1 would lead to a prolongation of the APD20 and APD50 of 25 and 27%, respectively. This result can be understood considering that the decrease in ICaL produced by CAF reduces its role in shaping the AP. Moreover, the small increase in the plateau height and duration produced dynamic modifications in the IKr and IKs amplitudes which combined with the NO-induced IKr block and IKs increase led to a shortening in the APD90.


Figure 6
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  		Figure 6 Mathematical modelling of human atrial action potentials in normal conditions (sinus rhythm, A) and in patients with chronic atrial fibrillation (70% reduction in ICaL and 50% in Ito1 and IKur, B) in the absence and after establishing the nitric oxide levels at 200 nM. The model in the presence of nitric oxide was run by incorporating the effects produced by nitric oxide on the currents shown in (C)–(F). The currents are the results of both the nitric oxide effects and the dynamic modifications that they produced on the action potential morphology.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
The present results demonstrated that (i) NO and NO donors (SNAP and DEANO) inhibited IKv4.3 and human atrial Ito1. (ii) NO inhibition was mediated by the activation of AC and the subsequent activation of PKA and PP2A. (iii) LAA from CAF patients presented the same NOS3 expression level as in SR patients. (iv) There was no difference in the Ito1 characteristics measured in ventricular myocytes from WT and NOS3–/– mice. (v) The increase in NO levels to 200 nM prolonged the plateau phase of the mouse atria, and lengthened the APD20 and APD50 of the human atrial CAF-modified AP as determined using a mathematical model.

4.1 Signalling pathways involved in the effects of nitric oxide on IKv4.3 and Ito1
NO produced a concentration-dependent inhibition of IKv4.3 which was reproduced in human atrial Ito1. The observation that the NO solution and two chemically dissimilar NO donors, whose only common denominator is their ability to release NO, inhibit IKv4.3 in a qualitatively and quantitatively similar manner suggests that this modulation is mediated by NO and not by the consequence of other NO-independent mechanisms.

The inhibition of Ito1 by NO was independent of the main pathways involved in NO effects in other substrates (GC/cGMP/PKG pathway, PKC activation, or redox-dependent post-translational modifications).2,17 The mechanism responsible for NO inhibition of IKv4.3 and human Ito1 was the activation of AC (which can be activated by NO through a mechanism that involves G proteins)21 and the subsequent activation of AC/cAMP/PKA pathway and PP2A. Other studies demonstrated that increased cAMP generation may be able to stimulate dephosphorylation of proteins. Indeed, an increase in cAMP levels can activate the PP2A by increasing the rates of PP2A methylation.18 Previous reports demonstrated that the activation of either the AC/cAMP/PKA pathway or the PP2A can inhibit human and rat Ito16,13,22 and that the AC/cAMP/PKA pathway is responsible for the NO inhibition of cardiac INa.20

The results suggest that the PKA phosphorylation and PP2A dephosphorylation take place in the Kv4.3 proteins and not in one of the ancillary β-subunits that form the native channel complex. Though it may be somewhat surprising that Kv4.3 is regulated by its simultaneous phosphorylation and dephosphorylation, this is not the first example of such a mechanism of regulation. Indeed, activation of NOS3 promoted by heat shock protein 90 involves its phosphorylation and dephosphorylation in two different residues.23 Similarly, PP2A dephosphorylation of the Kv4.3 protein probably takes place in a different residue than the PKA phosphorylation site, like the PKC or the PKG phosphorylation sites.22

4.2 Putative role of nitric oxide in electrical changes in chronic atrial fibrillation
Our results suggest that CAF does not modify NOS3 expression in human LAA. This is in agreement with the findings by Cai et al.,3 who demonstrated in a porcine model of AF that NOS3 expression in the LAA did not differ from that in control animals despite the reduction in NO formation at this site. However, in other parts of the atria, the authors did find a significant decrease in NOS3 expression. Moreover, there is another report using a canine model of AF demonstrating an increase in NOS3 expression.24 Thus, caution should be exerted in extrapolating the present finding to the whole atria. Finally, it has to be considered that the characteristics and/or the pharmacological treatment of the patients could also have determined the NOS3 expression level.

What is clearly established, both in patients and in animal models, is that NO bioavailability decreases in AF because of the increased atrial oxidative stress which scavenges NO to form the potent oxidant peroxynitrite and ‘uncouples’ NOS activity.4,5 This increase in the oxidative stress and decrease in NO concentrations may play an important role in the atrial oxidative injury and electrophysiological remodelling observed in CAF patients. The key role of NO in AF has also been suggested by the presence of a polymorphism in the NOS3 as a predisposing factor to AF.25

The present results demonstrated that prolonged decrease in NO concentrations does not modify the Ito1 density in NOS3–/– mouse myocytes. This result is similar to that previously reported for ICaL density.26 These authors used the same knockout strain as that used in the present experiments, in which it has been demonstrated that NOS3 suppression does not produce an upregulation of the neuronal and inducible isoforms of the NOS, and that the nitrite/nitrate levels were significantly decreased compared with WT.26,27 Recent reports stressed the important role of Kv4.2 alpha subunits (together with Kv4.3) in generating the Ito1 channels in the mouse heart.28 We also tested the effects of SNAP on IKv4.2 resulting in an inhibition of the current (30.4 ± 3.8%, n = 5, P < 0.05, not shown) not different from that produced on IKv4.3. Thus, it seems difficult to attribute the lack of effects of chronic NO deficiency on Ito1 to the heteromeric composition of the channel. Therefore, it seems reasonable to assume that the decrease of NO itself cannot account for the decrease in Kv4.3 expression observed in CAF patients.

Here we demonstrate that NO, at physiological concentrations (200–900 nM),29 inhibited human Ito1. In SR patients, AP is shaped by the presence of these physiological concentrations of NO, whereas in CAF patients, in which NO bioavailability is markedly decreased, the absence of NO could modify the AP characteristics. Thus, we analyse the effects of NO on CAF-modified AP by running a mathematical model previously validated for similar purposes,16 by incorporating all the described effects of NO in human atrial ionic currents. However, a limitation of this approach is that the effects of NO on each individual current were obtained under different experimental conditions (native currents and transfected cloned channels) and in healthy cells dissociated from hearts of different species. The model predicted that 200 nM NO would prolong the plateau phase of CAF-remodelled atrial AP. Furthermore, the plateau phase prolongation was confirmed when the effects of SNAP were analysed in multicellular preparations from WT and NOS3–/– mice. In this case, SNAP prolonged the APD20 by 25%, whereas it shortened both the APD50 and APD90. The discrepancy observed in the effects produced on the APD50 could be attributed to unknown effects produced by SNAP on outward K+ currents exclusively present in the mouse myocardium.

In human atria, the inhibition of IKur and Ito1 would increase the height and duration of the plateau,15 a prolongation that was critical for the termination of the periodic activity of sustained, high frequency, functional re-entrant sources (rotors), which could be responsible for AF.30 In fact, rotor termination was related to a prolongation of the APD at the plateau, rather than at the terminal phase of repolarization. In this respect, it has to be stressed that the combined inhibition of Ito1 and IKur11 produced by NO results in a prolongation of the plateau duration in human CAF-remodelled AP, which reached 83% at 30% of repolarization.

Overall, the results presented here suggest that the increase in atrial NO bioavailability could prolong the duration of the plateau phase of CAF-remodelled AP by inhibiting the Ito1 as a result of the activation of AC, which in turn activates PKA and PP2A. Therefore, the presence of physiological NO concentrations in the human atria could contribute to the prevention and/or cessation of the AF by the modulation of Ito1 and IKur. Thus, it is tempting to speculate that drugs that increase NO availability may exert a beneficial effect in patients with CAF. In fact, interventions that prevent activation of NADPH oxidases by vasoactive hormones or pro-inflammatory cytokines (i.e. angiotensin-converting enzyme-inhibitors, angiotensin II receptor blockers, or statins) are highly effective in preventing the occurrence of AF in patients with hypertension, heart failure or after heart surgery.1


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
Supported by Ministerio de Educación y Ciencia (SAF2005-04609), Ministerio de Sanidad y Consumo, Instituto de Salud Carlos III (Red HERACLES RD06/0009), Sociedad Española de Cardiología, and Fundación LILLY. Ricardo Gómez is a fellow of Comunidad Autónoma de Madrid.


   Acknowledgements
 
We thank Guadalupe Pablo for her excellent technical assistance and Drs T. Pérez-García and J.R. López-López (School of Medicine, Valladolid University, Spain) for the generous gift of the Kv4.2 cDNA.

Conflict of interest: none declared.


   Notes
 
{dagger} These two authors contributed equally. Back


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 

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