Peroxynitrite formation mediates LPC-induced augmentation of cardiac late sodium currents

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Journal of Molecular and Cellular Cardiology 44 (2008) 241–251www.elsevier.com/locate/yjmcc

Original article

Peroxynitrite formation mediates LPC-induced augmentationof cardiac late sodium currents

Mathieu Gautier a, Henggui Zhang b, Ian M. Fearon a,⁎

a Faculty of Life Sciences, The University of Manchester, Floor 2, Core Technology Facility, 46 Grafton Street, Manchester, M13 9NT, UKb Biological Physics Group, School of Physics and Astronomy, The University of Manchester, Manchester, UK

Received 18 June 2007; received in revised form 31 August 2007; accepted 10 September 2007Available online 21 September 2007

Abstract

Lysophosphatidylcholine (LPC) accumulates in the ischaemicmyocardium and is arrhythmogenic.We have examined themechanisms underlyingthe effects of LPC on the late cardiac Na+ current (ILNa). Na

+ currents were recorded in HEK293 cells expressing NaV1.5 and isolated rat ventricularmyocytes. LPC enhanced recombinant ILNa, while it reduced peak Na

+ current. Computer modeling of human ventricular myocyte action potentialspredicted a marked duration prolonging effect and arrhythmogenic potential due to these effects of LPC on peak and late currents. Enhancement ofrecombinant ILNa was suppressed by the antioxidant ascorbic acid and by the NADPH oxidase inhibitor DPI. Inhibitors of the mitochondrial electrontransport chain (rotenone, TTFA and myxothiazol) were without effect on LPC responses. The superoxide donor pyrogallol was without effect onILNa. Enhancement of ILNawas abrogated by theNOS inhibitors L-NAME and 7-nitroindazole, while LPC induced an L-NAME-sensitive productionof NO, measured as enhanced DAF-FM fluorescence, in both HEK293 cells and ventricular myocytes. Despite this, the NO donors SNAP and SNPcaused no change in ILNa. However, SNAP enhanced TTX-sensitive recombinant and native ILNa in the presence of pyrogallol, suggestingperoxynitrite formation as a mediator of the response to LPC. In support of this, the peroxynitrite scavenger FeTPPS prevented the response of ILNa toLPC. Peroxynitrite formation provides a novel mechanism by which LPC regulates the late cardiac Na+ current.© 2007 Elsevier Inc. All rights reserved.

Keywords: Sodium channel; Late sodium current; NaV1.5; Nitric oxide; Superoxide; Peroxynitrite; NADPH oxidase

1. Introduction

Voltage-dependent Na+ channels are key elements in theinitiation and propagation of action potentials in excitable cells.In response to supra-threshold membrane depolarization, Na+

channels open and allow passage of Na+ ions from the cellexterior to the interior. This causes the rapid action potentialupstroke, which ceases due to a diminished electrostatic drivingforce for Na+ entry and fast inactivation of the Na+ channel

Abbreviations: LPC, lysophosphatidylcholine; HEK, human embryonickidney; ILNa, late Na+ current; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein; TTFA, thenoyltrifluoroacetone; SNAP, S-nitroso-N-acetyl-penicillamine; SNP, sodium nitroprusside; L-NAME, N (G)-nitro-L-argininemethyl ester; DPI, diphenyleneiodonium; TTX, tetrodotoxin; ROS, reactiveoxygen species; X/XO, xanthine/xanthine oxidase; NO, nitric oxide; NOS, nitricoxide synthase; NOX, NADPH oxidase.⁎ Corresponding author. Tel.: +44 0 161 275 5496; fax: +44 0 161 275 5600.E-mail address: [email protected] (I.M. Fearon).

0022-2828/$ - see front matter © 2007 Elsevier Inc. All rights reserved.doi:10.1016/j.yjmcc.2007.09.007

itself [1]. Following the upstroke, further action potential cha-racteristics depend on the transmembrane fluxes of K+ and Ca2+

ions through voltage-gated channels, and in cardiac cells Ca2+

entry is a major contributor to the plateau phase which ensuressustained myocardial contraction. Both the initial rapid de-polarizing and prolonged plateau phases of the action potentialare also affected by a slowly inactivating or ‘late’ inward Na+

current [2]. Enhancement of this current leads to a dramaticalteration in Na+ loading, contributing to arrhythmias includingearly and delayed afterdepolarizations [2,3].

The pore-forming (α1) subunit of the voltage-dependent Na+

channel is a ∼260 kDa protein which, when expressed in theabsence of auxiliary (β) subunits, gives rise to a functionaldepolarization-activated Na+ channel [1]. To date, 9 α subunitshave been cloned from human tissue (termed NaV1.1, NaV1.2and so on; [4]), which despite their high sequence homologyexhibit markedly different functional properties. The predom-inant cardiac α isoform, NaV1.5, is encoded by the humanSCN5A gene [2,5–7]. A number of gain-of-function inheritable

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http://dx.doi.org/10.1016/j.yjmcc.2007.09.007

242 M. Gautier et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 241–251

mutations in the SCN5A gene impair Na+ channel inactivationand give rise to the ‘channelopathy’ LQT3 [8]. This arrhyth-mogenic impairment of inactivation allows persistent Na+ entryduring depolarization and prolongs the cardiac action potential[7].

Lysophosphatidylcholine (LPC) is formed by the hydrolysisof the membrane phospholipid phosphatidylcholine by the ac-tion of phospholipase A2 [9]. LPC accumulates during myocar-dial ischaemia and is arrhythmogenic [10–15], with effects onNa+ channel gating playing a major role in arrhythmogenesiseffects [16–20]. Here, we report that prolongation of the open-ing of cardiac Na+ channels is mediated by LPC-induced pero-xynitrite production, formed secondary to the production andreaction of superoxide and NO, shedding new light onto themechanisms involved in LPC-induced arrhythmogenesis.

2. Materials and methods

2.1. Cell culture

Experiments on recombinant Na+ channels were carried outin HEK293 cells stably expressing the hNaV1.5 α subunit. Thiscell line was kindly provided by Dr. J. Makielski (Departmentof Medicine and Physiology, University of Wisconsin, Madi-son). Construction of this stable line was described previously[21]. Cells were grown in minimum essential medium withEarle's salts and L-glutamine (Invitrogen), containing 9% (v/v)fetal calf serum (Globepharm), 1% (v/v) non-essential aminoacids, gentamicin (50 mg l−1), 10000 μl−1 penicillin G, 10 mgl−1 streptomycin and 0.25 mg l−1 amphotericin (all Invitrogen)at 37 °C in a humidified atmosphere of air/CO2 (19:1). Cellswere split twice weekly at a ratio of 1:5 and plated in 35 mmculture dishes for further culture or for use in experiments.

2.2. Electrophysiology

Dishes with attached cells were transferred to a continuallyperfused recording chamber and whole-cell patch-clamp record-ings [22] were made using patch pipettes of resistance 4–5 MΩ.Cells were perfused with the following (in mM): NaCl, 140;KCl, 4; CaCl2, 1.8; MgCl2, 0.75; HEPES, 5; and glucose, 10 (pH7.4 with NaOH). Patch electrodes were filled with (mM): CsF,120; CsCl, 20; EGTA, 2; and HEPES, 5 (pH 7.2 with CsOH).Traces were filtered at 5 kHz and digitized at 10 kHz. Capa-citative transients were minimized by analogue means (residualtransients have been truncated for illustrative purposes). Ana-lyses and voltage protocols were performed using a Multiclamp700A amplifier in combination with a Digidata 1322A andpCLAMP 9.0 software (all Axon Instruments). Recordings weremade at room temperature (22±2 °C).

2.3. Measurement of NO production

In experiments with HEK293 cells, these were seeded ontoglass coverslips 24 h prior to experiments. Cells were loadedwiththe membrane-permeant NO indicator dye, 4-amino-5-methyla-mino-2′,7′-difluorofluorescein (DAF-FM) diacetate (10 μM) in

HEPES-buffered saline at room temperature for 20 min. Sub-sequently, cells were incubated with dye-free HEPES-bufferedsaline for a further 20 min to allow de-esterification. To monitorNO levels in isolated ventricular myocytes, cells were loadedwith 20 μM DAF-FM in HEPES-buffered saline for 10 min atroom temperature, followed by a 10-min de-esterification. Dur-ing the initial incubation, myocytes adhered to a glass coverslip.For both cell types, slips were mounted on the stage of anOlympus IX71 inverted microscope and perfused with the samesalt solution used in electrophysiological studies. Individual cellswere selected for study using a manual diaphragm (Cairn). Thedye was excited at a wavelength of 480±40 nm using a BurleighExfo mercury light source and appropriate filtering (ChromaTechnology Corporation, Rockingham, VT, USA). Emitted lightwas passed through a 505-nm long-pass dichroic filter and col-lected at 535±50 nm (Chroma). The dye was excited for a periodof 100 ms every 2.5 s. Emitted light was detected using aphotomultiplier tube (Cairn). Amplified signals were digitizedusing a Digidata 1322A interface and recorded using pCLAMP9.0 software.

2.4. Isolation of ventricular myocytes

Myocytes were isolated from the ventricles of 4- to 6-week-old male Wistar rats, as previously described [23]. In brief, ratswere killed by stunning and cervical dislocation. After isolation,hearts were perfused through the aorta with Ca2+-free HEPES-buffered solution (HBS) to clear the heart of blood. After 1–2 min, 0.2 mg ml−1 collagenase and 25 μM Ca2+ were added tothe perfusate and the heart was continually perfused for 30 min.After this, the heart was chopped with fine scissors and thetissue triturated to release cells. These were gently centrifuged,then resuspended and stored in HBS containing 250 μM Ca2+.Cells were used in electrophysiological studies 2–3 h afterisolation. Care and use of animals were in accordance with theAnimals (Scientific Procedures) Act 1986, which conforms toNIH guidelines.

2.5. Computer modelling of human ventricular myocyte actionpotentials

The human ventricular cell model developed by ten Tusscheret al. [24] was used in this study. This was chosen as it incor-porates available human ventricular cell and membrane channeldata and reproduces known transmural APD andAPD restitutionbehaviour for epicardial (EPI), mid-myocardial (MIDDLE) andendocardial (ENDO) action potentials [24]. Recently, we havemodified the model to include the late sodium current (ILNa)[25]. To simulate the effects of LPC on Na+ currents, the channelconductance and inactivation time constant were modified toreproduce the experimental data of Ipeak reduction and ILNaincrease as shown in Fig. 1A. Modeling was run at 37 °C.

2.6. Chemicals and statistical analyses

LPC, rotenone,myxothiazol, TTFA, ascorbic acid, SNAP, SNP,pyrogallol, xanthine, xanthine oxidase, L-NAME, 7-nitroindazole

Fig. 1. LPC potentiates late and inhibits peak Na+ currents. Currents were recorded in HEK293 cells stably expressing hNaV1.5. (A) Typical current records obtainedbefore and following exposure to 5 μMLPC via the extracellular perfusate, as indicated. Currents were evoked by step depolarizing cells from −140 to −40 mV. Arrowindicates Ipeak in the presence of LPC. (B and C) Representative time series recordings demonstrating effects of LPC on late (ILNa; B) and peak (Ipeak; C) Na

+ currents.Currents were evoked as in panel A at a frequency of 0.1 Hz. Data were normalized to those at the start of the recording (I/I0). ILNa was measured 10 ms following thestep depolarization, as indicated by the vertical grey bar in panel A. LPC was applied as indicated by the horizontal bars. (D) Typical recordings demonstratingnormalized ILNa and Ipeak in cells perfused with an LPC-free solution. Inset, current traces taken from points in the time series recording indicated by vertical arrows.(E) Mean (±S.E.M.) data derived from recordings such as those shown in panels B and C. Data were averaged from the normalized current evoked before (control) andafter a 6-min exposure to LPC, from 8 cells (ILNa) and 8 cells (Ipeak). ⁎, Pb0.05. (F) Predicted effects of LPC on the mid-myocardial (MIDDLE) ventricular myocyteaction potential. Computer modeling was based on the models of ten Tusscher et al. [24], which were modified to incorporate ILNa. Data were modeled using traces asin panel A, under control conditions and in the presence of LPC as indicated. (G) Heterogeneous effects of LPC in epicardial (EPI), middle layer (MIDDLE) andendocardial (ENDO) myocytes. Action potential duration at 90% repolarization (APD90) and spatial APD dispersion between the indicated regions were obtained fromaction potential models such as those in panel F.

243M. Gautier et al. / Journal of Molecular and Cellular Cardiology 44 (2008) 241–251

andDPIwere from Sigma. FeTPPSwas fromMerckBiosciences(Nottingham, U.K.). DAF-FM diacetate was from Invitrogen.LPC was applied via the extracellular perfusate. When usingascorbic acid, DPI or NOS inhibitors, cells were incubated for1 h prior to recording Na+ currents and the drugs were alsoincluded in the extracellular perfusate. Results are expressed asmeans±S.E.M. Statistical comparisons were made usingStudent's paired or unpaired t-tests, as appropriate.

3. Results

3.1. LPC modulates recombinant Na+ currents

In HEK293 cells, stably expressing human NaV1.5 and inaccordance with previous studies [16–20], LPC, at a concen-tration attained early during cardiac ischaemia [5 μM; 26]enhanced late Na+ currents (ILNa) while it reduced peak Na+


currents (Ipeak; Fig. 1A). As exemplified in Fig. 1B, duringapplication of LPC via the extracellular perfusate, normalized(I/I0) ILNa steadily increased. Mean (±S.E.M.) normalized ILNawas 2.53±0.58 (n=8) after a 6-min exposure to LPC. This wassignificantly different to that in cells with control (LPC-free)solution (0.99±0.13; n=8; Pb0.05; Fig. 1D and E). As illus-trated in Fig. 1C, LPC caused a steady decrease in normalizedIpeak. Mean (±S.E.M.) normalized Ipeak values, averaged from8 cells, were 0.99±0.02 before and 0.63±0.06 after LPC ex-posure. Mean values following perfusion with an LPC-freeperfusate were 0.99±0.02 (n=8; Pb0.05 compared to cellsexposed to LPC; Fig. 1D and E).

To determine the effects of these changes in peak and lateNa+ currents, we used computer models of human ventricularmyocytes [24] and examined action potential characteristics.LPC is predicted to cause significant action potential prolon-gation (e.g. Fig. 1F), increasing the APD90 (Fig. 1F and G). Thiseffect was heterogeneous across EPI, MIDDLE and ENDOmyocytes, with the largest effect seen in the MIDDLE cellmodel (Fig. 1G). This effect caused spatial dispersion of actionpotential duration across the ventricular wall (Fig. 1G).

3.2. Involvement of ROS in response of late Na+ current to LPC

In many cells, effects of LPC are mediated by reactiveoxygen species (ROS) production [27–30]. To explore a role for

Fig. 2. Ascorbic acid blocks effect of LPC on ILNa. (A) Example recording showing t(200 μM). Cells were pretreated for 1 h prior to recording Na+ currents, and thedemonstrating inhibition of Ipeak by LPC in the presence of ascorbic acid. In panels Afrequency of 0.1 Hz. Data were normalized (I/I0). Late currents were measured 10 msTime series recordings in panels A and B were obtained in the same cell. (C) Individpresence of ascorbic acid. (D and E) Mean (±S.E.M.) data obtained from recordings sindicated in parentheses. ⁎, Pb0.05. NS, PN0.05.

ROS in the LPC-mediated modulation of recombinant Na+

currents, we examined responses to LPC in the presence of theantioxidant ascorbic acid. In experiments where cells werepretreated with 200 μM ascorbic acid and in which the anti-oxidant was also present during the recording, enhancement ofILNa in response to LPC was prevented (Fig. 2A, C and D).Mean (±S.E.M.) normalized ILNa in the presence of ascorbicacid was 0.98±0.07 prior to, and 1.21±0.08 subsequent to,exposure to LPC (n=7 in each case). The latter mean value wassignificantly different from that seen in cells exposed to LPC inthe absence of the antioxidant (2.53±0.58; n=7; Pb0.05). Incontrast to this ablation of enhancement of ILNa, ascorbic aciddid not alter the inhibition of Ipeak by LPC (Fig. 2B, C and E).Thus, baseline normalized Ipeak in the presence of ascorbic acidwas 0.99±0.02, and 0.60±0.06 when cells were exposed toLPC (n=8 in each case). The latter mean value was not signi-ficantly different to that seen when cells were exposed to LPC inthe absence of ascorbate (0.63±0.06; n=7; PN0.05). Thesedata suggest that LPC causes its effects on peak and late Na+

currents by different mechanisms, with the effect on ILNa in-volving cell oxidant production.

To probe the source of LPC-induced oxidants, we initiallyutilized blockers of the mitochondrial electron transport chain(ETC), a source of LPC-induced ROS production [27,30].Neither the complex I inhibitor rotenone (1 μM), the complex IIinhibitor TTFA (10 μM) or the complex III inhibitor myxothiazol

he lack of effect of LPC on ILNa in the presence of the antioxidant, ascorbic acidantioxidant was also included in the perfusate. (B) Representative recordingand B, currents were evoked by step depolarizing cells from −140 to −40 mVat afollowing step depolarization. LPC was applied as indicated by horizontal bars.ual current traces demonstrating the lack of effect of LPC on Na+ currents in theuch as those shown in panels A and B. Data were obtained from number of cells


(500 nM) affected responses to LPC (Fig. 3A–C and E). Mean(±S.E.M.) normalized ILNa following exposure to LPC were2.18±0.35 (controls; n=7), 2.47±0.28 (rotenone; n=7;PN0.05 compared to LPC alone), 1.83±0.21 (TTFA; n=7;PN0.05) and 2.14±0.27 (myxothiazol; n=7; PN0.05). Wefurther examined the potential of NADPH oxidase as a source ofLPC-induced ROS production [31–33]. The NADPH oxidaseinhibitor DPI prevented the LPC response (Fig. 3D and E).Mean(±S.E.M.) normalized ILNa following exposure to LPC in thepresence of 5 μM DPI was 1.16±0.16 (n=5), not significantlydifferent from that seen in cells perfused with DPI alone (1.00±0.09, n=5; PN0.05) and significantly different from that due to

Fig. 3. Stimulation of NADPH oxidase mediates enhancement of ILNa by LPC. (A, Binhibitors of the mitochondrial ETC. Effects of LPC on ILNa were recorded with 1 μMperfusate. LPC was applied as indicated by horizontal bars. Currents were evoked(D) Representative recording demonstrating that the NADPH oxidase inhibitor, DPI (5above. (E) Mean (±S.E.M.) data obtained from recordings such as those in panels A–compared to LPC alone. NS, PN0.05 compared to LPC alone. (F and G) Mean (±S.xanthine/xanthine oxidase (500 μM/10 mU mL−1) or pyrogallol (100 μM). Periods oabove. Currents were normalized (I/I0) and averaged from 9 cells (xanthine/xanthine

LPC in the absence of DPI (2.18±0.35, n=7; Pb0.05). Thus,NADPH oxidase is a source of ROS production induced by LPCand which mediates the response of the Na+ channel to the lipid.

To further probe the role of ROS in the LPC-induced increasein ILNa, we recorded these currents while exposing cells to thesuperoxide-generating systems xanthine/xanthine oxidase (X/XO; 500 μM/10 mUmL−1) and pyrogallol (100 μM). As shownin Fig. 3F and G, neither system altered the magnitude of ILNa.Thus, after exposure normalized ILNa was 1.00±0.06 (n=9) incells perfused with X/XO and 1.15±0.05 (n=8) in cells perfusedwith pyrogallol. In each case, these values were not significant-ly different from those seen prior to oxidant system exposure

and C) LPC enhanced ILNa (presented as normalized data, I/I0) in the presence ofrotenone (A), 10 μM TTFA (B) or 500 nM myxothiazol (C) in the extracellularby step depolarizing cells from −140 to −40 mV at a frequency of 0.1 Hz.μM), prevented the response of ILNa to LPC. Currents were evoked as describedD. Data were obtained from number of cells indicated in parentheses. ⁎, Pb0.05E.M.) recordings of ILNa during exposure to the superoxide generating systemsf application are indicated by horizontal bars. Currents were evoked as describedoxidase) and 8 cells (pyrogallol).


(0.99±0.02 and 0.96±0.03 respectively; PN0.05 in each case).Together with the data obtained using antioxidants, these datasuggest that while oxidants are involved in the response of theILNa to LPC, these reactive species alone do not mediate theresponse.

3.3. A role for NO production in responses to LPC?

Previous studies demonstrated stimulation of the endothe-lial isoform of nitric oxide synthase (eNOS) by oxLDL andLPC [34,35]. To examine NO production as a potential mediator

Fig. 4. Potential involvement of NO in responses to LPC. (A and B) Example time s5 μM LPC was prevented by the NOS inhibitors L-NAME (500 μM; A) or 7-nitroincells stably expressing hNaV1.5 from −140 to −40 mVat a frequency of 0.1 Hz. (C)and B. Data were averaged from the normalized current evoked after a 6-min expoobtained in cells exposed to LPC (e.g. Fig. 1B) in the absence of NOS inhibition. ⁎

fluorescence of the NO indicator dye, DAF-FM, during exposure of HEK293 cells toNAME (□). When LPC was applied to cells, this occurred at the point indicated by threcordings such as those in panel D. Fluorescence intensities were obtained at the enindicated in parentheses. (F and G) Example recordings demonstrating the lack of effCurrents were evoked as in panel A. NO donors were applied as indicated by horizonin panels F and G. Data were obtained from the number of cells indicated in parenth

of the LPC response, we used the NOS inhibitors L-NAME(500 μM) and 7-nitroindazole (7-NI; 300 μM). Both inhibitorsfully blocked the enhancement of ILNa during exposure to LPC(Fig. 4A–C). In further support of a role for NO production,LPC induced an L-NAME-sensitive increase in fluorescencein cells loaded with the NO indicator dye, DAF-FM (Fig. 4Dand E). Mean (±S.E.M.) normalized (F/F0) DAF-FM fluores-cence following exposure to LPC was 1.20±0.05 (n=10) inthe absence and 1.07±0.01 (n=8) in the presence of 500 μML-NAME (Pb0.05). In control cells, fluorescence was 1.04±0.03 (n=10).

eries recordings demonstrating that the increase in normalized (I/I0) ILNa due todazole (7-NI, 300 μM; B). Currents were evoked by step depolarizing HEK293Mean (±S.E.M.) data derived from recordings such as those shown in panels Asure to LPC. Numbers of cells are indicated in parentheses. Control data were, Pb0.05 compared to LPC alone. (D) Representative recordings showing thea drug-free perfusate (○), 5 μMLPC (●) and LPC in the presence of 500 μM L-e arrow. Values were normalized (F/F0). (E) Mean (±S.E.M.) data derived fromd of the recording. DAF-FM fluorescence was monitored in the number of cellsect of the NO donors SNAP (200 μM) and SNP (200 μM) on normalized. ILNa.tal bars. (H) Mean (±S.E.M.) data obtained from recordings such as those showneses. NS, PN0.05 compared to control cells (perfused with donor-free solution).


Despite the abolition of the response to LPC and the observedLPC-induced, L-NAME-sensitive NO production, exposure ofHEK293 cells to the exogenous NO donors S-nitroso-N-acetylpenicillamine (SNAP; 200 μM) and sodium nitroprusside(SNP; 200 μM) evoked no change in ILNa (Fig. 4F–H). Mean(±S.E.M.) ILNa values following exposure to NO donors were1.11±0.10 (SNAP; n=6) and 0.94±0.07 (SNP; n=6). In eachcase, these values were not significantly different (PN0.05) tothose obtained in control cells (0.99±0.13; n=8) Thus, similarto superoxide, while NO is involved in the response to LPC it isnot the sole mediator.

3.4. Peroxynitrite mediates the increase in ILNa due to LPC

Data above demonstrated roles for NO and superoxide inresponses to LPC. However, exogenously applied individually,these reactive species did not recreate the LPC response. Thus, weinvestigated whether peroxynitrite (ONOO−, the product of thereaction of NO and superoxide [36,37]) mediated the enhance-ment of ILNa by LPC. As shown in Fig. 5A, simultaneous ap-plication of SNAP (200 μM) and pyrogallol (100 μM) increased

Fig. 5. Role for peroxynitrite in the effects of LPC on ILNa. (A) Mean (±S.E.M.) recoILNa through hNaV1.5. Cells were exposed to either 200 μM SNAP (□; n=6) or 10n=8). Donor perfusion was initiated as indicated by the arrow. (B) Example recordinFeTPPS (50 μM). In panels A and B, currents were evoked by step depolarizing ce(±S.E.M.) data obtained from recordings such as those in panel B. Data were averaLPC, in the absence and presence of FeTPPS, as indicated. Numbers of cells indic

ILNa. After such exposure, normalized ILNa was 2.16±0.35(n=8), significantly different from that seen in cells exposed toeach donor individually (SNAP, 1.11±0.10 and pyrogallol, 1.15±0.05; n=6 and 8 respectively; Pb0.05 in each case). This sug-gested that the response to LPC was mediated by peroxynitriteproduction. The peroxynitrite-induced current was sensitive to1 μMTTX, indicative of an effect on ILNa and not an endogenouscurrent. ILNa current amplitudes were −11.7±3.2 pA/pF (con-trol), −16.4±4.6 pA/pF (SNAP+pyrogallol) and −6.5±1.4 pA/pF (SNAP+pyrogallol+TTX; Pb0.05 compared to those in theabsence of TTX; n=4 in each case). In support of the proposedrole for peroxynitrite, enhancement of ILNa by LPC was pre-vented by the selective peroxynitrite scavenger, FeTPPS (50 μM;Fig. 5B andC). Normalized ILNa following a 7-min application ofLPC was 2.43±0.43 (n=6) in the absence and 1.15±0.11 (n=4)in the presence of FeTPPS (Pb0.05). In further support, pero-xynitrite produced by the simultaneous application of SNAP andpyrogallol to isolated ventricular myocytes significantly increasedthe magnitude of ILNa (Fig. 6A and B), such that normalized(I/I0) ILNa was increased from 0.97±0.02 to 1.41±0.08 (n=5;Pb0.05). In these cells, LPC induced a robust production of NO,

rdings demonstrating effects of NO and superoxide donors on normalized (I/I0)0 μM pyrogallol (○; n=8) alone or SNAP and pyrogallol simultaneously (●;g showing the abolition of the response to LPC by the peroxynitrite scavenger,lls from −140 to −40 mV at a frequency of 0.1 Hz and normalized. (C) Meanged from the normalized current evoked 6 min after initiating the exposure toated in parentheses. ⁎, Pb0.05 compared to LPC alone.

Fig. 6. Peroxynitrite enhances native ILNa. (A) Representative recording demonstrating the effect of simultaneous application of 200 μM SNAP and 100 μMpyrogallol on ILNa in an isolated ventricular myocyte. Currents were evoked by step depolarizing cells from −140 to −40 mVat a frequency of 0.1 Hz and normalized(I/I0). Inset, representative traces from the time series recording, under control conditions and in the presence of SNAP and pyrogallol, as indicated. Traces have beentruncated for illustrative purposes. Total charge transfer during the step depolarization was 39.9 pC (control) and 50.4 C (SNAP+pyrogallol). (B) Mean (±S.E.M.) dataderived from recordings such as that shown in panel A. Data were averaged from 5 cells before (control) and following exposure to LPC. ⁎, Pb0.05 compared tocontrol. (C) Representative recordings of DAF-FM fluorescence during exposure of ventricular myocytes to 5 μMLPC in the absence (○) and presence (●) of 500 μML-NAME. LPC was applied as indicated by the horizontal bar. Values were normalized (F/F0). (D) Mean (±S.E.M.) data derived from recordings such as thoseindicated in panel D. Fluorescence intensities were obtained at the end of the recording. DAF-FM fluorescence was monitored in 5 cells in each case. ⁎, Pb0.05compared to LPC alone.


measured as an increase in DAF-FM fluorescence (Fig. 6C),which was L-NAME sensitive (Fig. 6C and D).

4. Discussion

Slowing of Na+ channel inactivation and enhancement of lateNa+ currents prolongs the action potential and promotes signi-ficant arrhythmias, such as early and late afterdepolarizations[2,3,38]. Previous reports documented the effects of the is-chaemic metabolite lysophosphatidylcholine on cardiac NaV1.5currents, with the lipid exerting opposing effects on late and peakNa+ currents [16–20]. Our computer modeling based on pre-vious models [24] demonstrated that despite these differenteffects on peak and late currents, the overall effect of LPCwas toincrease action potential duration and spatial action potentialdispersion, which is suggested to be arrhythmogenic [24,39].

In the present study, we show that the effects of LPC on peakand late Na+ currents are mechanistically dissimilar. The re-sponse of the late, but not the peak, Na+ current to LPC wasprevented by the antioxidant ascorbic acid. Augmentation ofILNa by LPC was also blocked by inhibition of the superoxideproducing enzyme NADPH oxidase (NOX) and by inhibition ofnitric oxide synthases (NOS). In vitro measurement of NOproduction using the NO indicator dye DAF-FM demonstratedan L-NAME-sensitive, LPC-induced NO production in bothHEK293 cells and ventricular myocytes. The effects of LPC on

ILNa were mimicked by superoxide and NO donors appliedtogether (but not alone) and were blocked by selective peroxy-nitrite scavenging. Thus, enhancement of ILNa by LPC wasmediated by the peroxynitrite anion, formed secondary to theproduction and interaction of NOS-derived NO and NOX-derived superoxide [36,37]. LPC-induced peroxynitrite produc-tion provides a novel cardiac signalling paradigm and providesfresh information concerning the mechanistic basis of LPC-induced altered cardiac ion channel function and arrhythmia.Interestingly, a recent study showed that combined pretreatmentwith NO donors and antioxidants decreased myocardial injuryand reduced dityrosine formation, to a degree greater than thatseen with NO donation alone. This suggests that the beneficialeffects of NO and scavenging were mediated by reducing theformation of peroxynitrite during ischaemia/reperfusion [40],which is in accordance with our findings.

In many tissues and cell types, including vascular endothelialand smooth muscle cells, LPC and oxidized LDL (oxLDL; ofwhich LPC is the principal active component; [41]) regulateboth NOX and NOS to produce superoxide and NO respectively[31–33,42,43]. This combination of enzyme activation andsubsequent peroxynitrite formation has long-term effects, suchas mediating the insulin resistance of endothelial cells [44].However, our data implicate, for the first time, the product of thereaction between superoxide and NO in an acute response toLPC. In support of this rapid response to LPC, biochemical


assays showed that oxLDL stimulated eNOS acutely [34]. Fur-ther, LPC-induced ROS production by NADPH oxidase inendothelial and vascular smooth muscle cells occurred oversimilar time periods to those studied here [29,45]. Importantly,the observed effects of LPC on cardiac ILNa in our acute studiesoccur at a concentration reached early in cardiac ischaemia [26].

Although both superoxide and NO are well-described regu-lators of ion channel activity, our data demonstrate that theirreaction product, and not these reactive species alone, regulateILNa. With respect to this role of ONOO−, our findings contrastwith those of Ahern et al. [46], who demonstrated that perfusionwith the NO donor SNP markedly enhanced ILNa in ventricularmyocytes. However, a confounding factor in the previous studyis the use of high concentrations of SNP [46]. At such levels,this NO donor inhibits the O2

− quenching enzyme superoxidedismutase [47], leading to elevated O2

− levels and the potentialfor ONOO− formation. Whether this is the case remains to bedetermined. However, other studies have shown, similar to ourfindings, that although NO regulates peak Na+ currents it alonedoes not induce a late Na+ conductance or alter inactivationkinetics, which would be expected should ILNa be activated[48]. Interestingly, and in further accordance with our workONOO−, but not NO itself, induced arrhythmias in numerousexperimental models [49].

In our studies, we have used a recombinant system to exa-mine responses to LPC and explored the underlying biochem-ical mechanisms of the increase in ILNa. Information gainedfrom these studies was further used in an isolated myocytepreparation to demonstrate the potential of peroxynitrite inenhancing late Na+ currents. Interestingly, while simultaneousSNAP and pyrogallol enhanced native ILNa, the magnitude ofthis effect was not as large as that seen for the recombinantchannel. While we have no demonstrated explanation for thisdifference, it may potentially arise due to an auxiliary (β; [1])subunit expressed in the native system modifying the responseto LPC. Of further potential is the cardioprotective ability ofcardiac myocytes to scavenge exogenous free radicals [50].Despite this difference, however, it is likely that even modesteffects of peroxynitrite on ILNA are arrhythmogenic. Our owncalculations show a marked increase in transmembrane Na+

charge movement following peroxynitrite exposure, while thestudies of Light et al. [38] demonstrate that activation of ILNa toa similar magnitude as that induced by LPC in our native cellsprolongs action potential duration and is pro-arrhythmogenic,causing early afterdepolarizations.

Concerning the role and source of ROS in LPC responses,we recently documented a ROS-mediated effect of LPC onnative and recombinant cardiac Ca2+ channels, in which longerterm (3–4 h) exposure of cells to LPC enhanced Ca2+ currentamplitudes secondary to ROS production within mitochondrialcomplex I [27]. Other studies also demonstrated similar altera-tions of mitochondrial function and ROS production by pro-longed LPC or oxLDL exposure [30,51,52]. However, in thepresent study examining short-term responses of the cardiacNa+ channel to LPC, pharmacological abrogation of the mito-chondrial ETC did not disrupt enhancement of ILNa. Previous-ly, we had used a genetic model in which mitochondrial DNA,

and subsequently function, was disrupted by long-term expo-sure to ethidium bromide [27,53]. Despite several attempts insetting up such a ρ0 cell line of HEK293 cells stably expressingNaV1.5, long-term exposure of these cells to ethidium bromideat concentrations known to ablate the ETC constantly resultedin cell death. Thus, we were unable to use this mitochondria-targeted approach to verify the lack of involvement of the ETC.However, the response was prevented by DPI, which is con-sistent with the lack of ETC involvement and suggestive of arole for the superoxide-producing plasmalemmal NOX enzymecomplex. These findings lead to the possibility that the source ofROS is dependent on the downstream effector system beingregulated. Acutely, LPC stimulates a plasmalemmal source ofROS which gives rise to altered currents through a transmem-brane protein, NaV1.5. The ETC, located in predominantlyperinuclear mitochondria [27] is thus not required as a mediatorof this response. Chronically, LPC causes mitochondrial (i.e.intracellular) ROS production, and the downstream effect isaltered trafficking of the ion channel, CaV1.2 [27].

In endothelial cells, both NOX and eNOS are found in aspecific membrane compartment, localized within caveolin-richdomains [54]. While a similar localization has not been demon-strated in cardiac myocytes, it is of interest that both eNOS andNaV1.5 are located within caveolin-rich domains in the heart[55–57]. Currently, there is no experimental evidence in theheart to suggest a localization of NOX either within or excludedfrom caveolin-rich domains. However, such a compartmental-ization of the channel and the NOX and eNOS enzymes wouldprovide an even more discrete localized domain in whichperoxynitrite could exert its effects on the Na+ channel. Furtherwork is necessary to examine this possibility by examining thecolocalization of these plasmalemmal proteins and complexes,potentially within caveolae.

In summary, peroxynitrite formation mediates the effects ofthe ischaemic metabolite LPC on cardiac ILNa. These findingsdescribe a potentially important signalling mechanism in theischaemic heart and provide new insight into arrhythmogenesisdue to LPC.

Acknowledgments

Experiments were supported by a Project Grant (PG/05/042)to IMF from the British Heart Foundation. Equipment wasprovided by grants from the British Heart Foundation and theRoyal Society. HEK293 cells stably expressing hNaV1.5 werekindly provided by Dr. J. Makielski (Department of Medicineand Physiology, University of Wisconsin, Madison). Ventricularmyocytes were provided by the Unit of Cardiac Physiology,Faculty of Medical and Human Sciences, The University ofManchester. We gratefully acknowledge the help and advice ofProfessor Mark Boyett (Unit of Cardiac Physiology, Faculty ofMedical and Human Sciences) with analysis and modelling.

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Peroxynitrite formation mediates LPC-induced augmentation of cardiac late sodium currents