Georges Christé

Effects of low [K + ] o , on the electrical activity of human cardiac ventricular and Purkinje cells

Keywords: action potentials, resting potentials, heart ventricle, Purkinje fibres, potassium, tetrodotoxin, arrhythmia

Ventricular and Purkinje action potentials were recorded with a microelectrode in a strip of human papillary muscle. Lowering the K-content of the superfusing solution from 5.9 to 0.5 mmol.litre -1 at 37°C hyperpolarised ventricular diastolic potential steadily as long as [K + ] o was low (up to 70 min tested). Ventricular action potentials were transiently lengthened and then shortened. A positive inotropic effect was noted and attributed to Na-K pump inhibition since it was reversed by the addition of 2 mmol.litre -1 thallous chloride to the low [K + ] o solution. Beyond 40 min, transient depolarisations and after-contractions were found. During the first minutes in low [K + ] o , Purkinje diastolic potential was hyperpolarised and the action potential was lengthened at all levels of repolarisation. Afterwards, the Purkinje diastolic potential suddenly depolarised by 30 mV. Restoration of the control solution caused a slow repolarisation and then a sudden return of the diastolic potential to near control value. This was reproduced during drive (38 stim.min -1 ) and at rest. At the depolarised level of potential, stimulation elicited slow action potentials with diastolic slow depolarisation and spontaneous oscillations of potential appeared at rest. In Purkinje cells, increasing concentrations of tetrodotoxin from 10 -7 to 8 X 10 -6 mol.litre -1 in the control solution shifted the diastolic potential in negative direction by a few mV and shortened the action potential duration at all levels of repolarisation. The possible implications of these phenomena in the genesis of some cardiac arrhythmias are discussed.

Purkinje fibres differ in their electrophysiological properties from ventricular myocardial fibres in mammals, [START_REF] Gettes | Effects of low and high concentrations of potassium on the simultaneously recorded Purkinje and ventricular action potentials of the perfused pig moderator band[END_REF] including man. [START_REF] Dangman | Electrophysiologic characteristics of human ventricular and Purkinje fibers[END_REF] Particularly, the duration of the action potential and the effective refractory period are longer in Purkinje than in ventricular myocardial fibres. These are longest in distal Purkinje fibres which act as a "gate" [START_REF] Myerburg | Electrophysiology of endocardial intraventricular conduction: the role and function of the specialized conducting system[END_REF] preventing the propagation of premature impulses. This phenomenon was termed the "gating mechanism" by Myerburg et al. [START_REF] Myerburg | Electrophysiology of endocardial intraventricular conduction: the role and function of the specialized conducting system[END_REF] The two tissues respond differently to several agents. When [K + ] o is lowered from about 5 to about 2 mmol.litre -1 , the duration of the action potential and the effective refractory period are lengthened in Purkinje fibres but shortened in ventricular myocardium. [START_REF] Gettes | Effects of low and high concentrations of potassium on the simultaneously recorded Purkinje and ventricular action potentials of the perfused pig moderator band[END_REF] Purkinje fibres may even be dramatically depolarised when [K + ] o is further lowered 4 5 while ventricular fibres are not. 6 7 However, Na-K pump blockade by digitalis or low [K + ] o induces transient depolarisations of both mammalian Purkinje [START_REF] Kass | Ionic basis of transient inward current induced by strophantidin in cardiac Purkinje fibres[END_REF] and ventricular fibres. [START_REF] Eisner | Inotropic and arrhythmogenic effects of potassium depleted solutions on mammalian cardiac muscle[END_REF] Agents that depress the sodium current (ie tetrodotoxin and local anaesthetics) also depress the steady-state ("window") sodium current 10 11 and shorten Purkinje action potentials considerably more than ventricular myocardial ones. 12 13 All these phenomena have been involved in the explanation of cardiac arrhythmias. [START_REF] Cranefield | The conduction of the cardiac impulse[END_REF] During a study of inotropic and arrhythmogenic effects of Na-K pump blockade by low [K + ] o , we recorded strikingly different effects between myocardial and Purkinje fibres in a strip of human ventricle. Our results are quite similar to well established data from mammals and may be relevant to the mechanism of cardiac arrhythmias due to Na-K pump poisoning.

Methods

During surgical insertion of a valvular prosthesis, part of a papillary muscle was removed from the right ventricle of a 6 year-old male patient with a tetralogy of Fallot. This excision was part of the operative procedure, the excised part being discarded if unused for experiments. The patient had been receiving 8 mg.kg -1 (body weight) digoxin daily for 5 years.

Upon excision, the sample was dropped into a solution at room temperature (20°C) having the following salt composition (mmol.litre -1 ): NaCl 117, KCl 3.1, KH 2 PO 4 1.2, NaHCO 3 24.8, CaCl 2 2.5, MgS0 4 1.2, glucose 11.2 in distilled water and continuously gassed with CO 2 5% in O 2 . [START_REF] Krebs | Untersuchungen über die Hamstoftbildung im Tierkorper[END_REF] This was the control solution used in the experiments.

Transport to the laboratory took 10 min. A strip (2 x 1 x 1 mm) was then cut with scissors and pinned onto the sylgard-covered bottom of a 1.5 cm 3 perspex bath. Superfusion was started at a constant rate of 3 cm 3 .min -1 (*) for 30 min (peristaltic pump LKB 2115)) the temperature being elevated to 37°C within the first 10 min.

(*) Absolute delay from switch to input of bath and times needed for 99% rise and decay of bath [Na + ] between 0 and 140 mmol.litre -1 were evaluated to be 1.7 min, 6.5 min and 10 min respectively.

During another 30 min stabilisation period and during experiments, stimulations at 1.5 times threshold magnitude (dc square pulses 20 mA, 1.5 ms) were applied through an isolating unit between a large electrode in the bath and a punctate one near one end of the preparation. Both were silver-silver chloride electrodes. Unless otherwise stated, the driving frequency was 38 stim.min -1 .

Microelectrodes (resistance 30 to 50 MΩ) were filled with 3 mol.litre -1 potassium chloride acidified to pH 2 with added HCl in order to reduce the tip potential. [START_REF] Riemer | Determination of the membrane potential in smooth muscle using microelectrodes with reduced tip potential[END_REF] The mean tip potential value in five microelectrodes was 5±1 mV. A microelectrode was mounted in a semi-floating fashion [START_REF] Sato | Modifications of glass microelectrodes: a self-filling and semi-floating glass microelectrode[END_REF] on a Narishige micromanipulator. It was brought near the preparation, but kept away from damaged regions, and a stable impalement was searched for. The results shown below were obtained during impalements which remained stable during a control period of at least 10 min and for at least 15 min after changing the solution. Each figure was constructed from records obtained during a single impalement, unless otherwise stated.

Potentials were recorded through a high input impedance follower amplifier built with operational amplifiers (input impedance 10 12 Ω, input current 10 pA) with a gain of 10. The reference electrode in the bath and the connection to the microelectrode were both silver-silver chloride electrodes.

Chart records (Gould Brush 2400) and pictures of the oscilloscopic display were taken (Tektronix RM 565 oscilloscope and Nihon-Kohden PC2A camera).

The modified K-depleted solution was prepared by uncompensated omission of all the KC1 and replacement of the appropriate amount of KH 2 P0 4 by NaH 2 PO 4 . Tetrodotoxin (TTX) (Crystalline 3X Sankyo) was diluted into an aliquot of the control solution to 10 -5 mol.litre -1 . Appropriate amounts of this solution were added to the control solution from the same batch to give the final concentrations given in the text.

Results

Two different action potential configurations were found in the preparation in control solution (fig 1). Some action potentials were very similar to the ones recorded by others in ventricular myocardial cells of adult humans. 2 7 17 Purkinje action potentials were also recorded. They showed initial rapid repolarisation and a low plateau (fig 1) and looked similar to action potentials recorded in Purkinje cells of adult man 2 and monkey. [START_REF] Gautier | The site of gating in the ventricular conducting system of rabbit, dog and monkey hearts[END_REF] Their duration at the late repolarisation phase was about 20% longer than in ventricular action potentials (fig 1). These effects are in agreement with reported ones in ventricular myocardial preparations from mammals (eg 1 19 ) including man [START_REF] Prasad | Transmembrane potential and contraction of human heart in relation to potassium and ouabain[END_REF] and amphibian. ). Such inotropic and arrhythmogenic effects can be attributed to inhibition of the Na-K pump in low [K + ] o . 9 21 23 Thallous ions (2 mmol.litre -1 thallous chloride) were added to the 0.5 mmol.litre -1 solution in order to replace 3 mmol.litre -1 K + ions for the activation of the Na-K pump and to reverse related effects of low [K + ] o . [START_REF] Eisner | The relationship between sodium pump activity and twitch tension in cardiac Purkinje fibres[END_REF] In A and B driving rate: 20 stim.min -1 .

FIG 2 Modifications of ventricular myocardial action potential during exposure to low [K + ] o . A: Slow chart record. Note progressive hyperpolarisation of the diastolic membrane during the first 10 min. A slower hyperpolarisation followed at later times (eg at 40 min). B: Superimposed action potentials (redrawn from film images of the oscilloscope screen) obtained at times shown by the arrows labelled "a" to "d" in A. Note shortening of plateau duration and transient lengthening of final repolarisation at 4 min (b), and uniform shortening of action potential duration at 9 min (c). Beyond 30 min (eg (d) at 40 min), the late repolarisation phase was lengthened.

DIASTOLIC AND ACTION POTENTIAL CHANGES IN LOW [K + ] o

Myocardial ventricular cells

FIG 5 Driven human Purkinje cell. A: Slow chart record of the diastolic potential during exposure to low [K + ] o , and after returning to control solution. B: Superimposed action potentials (redrawn from film images of the oscilloscope screen). a: in control solution; b and c: in low [K + ] o at times shown by the foot of each oblique arrow in A. C: Fast chart record of action potentials triggered at the low level of potential. Note the slow rising phase and the diastolic depolarisation. Initial sharp spike corresponds to stimulation artefact.

When the preparation was not stimulated, fairly similar phenomena were recorded (fig 6). The resting potential was stable in the control solution at about -80 mV. Superfusion with the K-depleted solution (fig 6) caused a progressive hyperpolarisation as when stimulated. After a few minutes, a depolarisation started which gave rise to an action potential seen as a sharp spike in fig 6. The repolarisation phase of this action potential was not complete: after a downward "notch", the potential stabilised around -45 mV. In one case, spontaneous oscillations appeared with a proper frequency around 1 Hz (see insert to trace B of fig 6). Restitution of the control solution caused a slow repolarisation to -60 mV in 1 min and then the resting potential suddenly shifted to a value slightly more negative than the control value. Afterwards it slowly returned to control.

FlG 6 Effects of lowering [K + ] o , and of returning to control solution on the resting potential of two human cardiac Pukinje cells. In A and B, the upper thick line denotes time of low [K + ] o , superfusion, middle trace is the membrane potential and the lower thin line shows clock pulses:

short every minute, long every 10 min.

EFFECTS OF TTX ON PURKINJE ACTION POTENTIAL

The preparation was exposed to gradually increasing concentrations of TTX from 10 -7 to 8 x 10 -6 mol.litre -1 by successive additions of stock TTX solution to the control solution within a 40 min period. The microelectrode remained impaled in the same Purkinje cell during the whole experiment. Fig 7 shows that TTX shifted the diastolic potential in a negative direction. The hyperpolarisation was maximal with 5 x 10 -6 mol.litre -1 TTX and for higher concentrations it slightly decreased. TTX also decreased action potential duration at all levels of repolarisation. The higher the concentration, the greater the shortening.

Discussion

The shape of ventricular action potentials recorded in the present preparation and the value of the diastolic potential in the control solution (eg fig 1) were similar to the ones reported from adult human ventricular myocardium. 2 7 17 The preparation thus behaved as normal healthy myocardium, and seemed undisturbed by the previous digoxin treatment.

The finding of Purkinje action potentials (fig 1) in a papillary muscle may be attributed to subendocardial conductive cells. 24 25 DIASTOLIC POTENTIAL CHANGES IN LOW [K + ] o In ventricular cells, low [K + ] o hyperpolarised the membrane, even after long durations (fig 2). This is in agreement with previous results of papillary muscles from adult human [START_REF] Prasad | Transmembrane potential and contraction of human heart in relation to potassium and ouabain[END_REF] and guinea-pig [START_REF] Reiter | Sodium dependence of the inotropic effect of a reduction in extracellular potassium concentration[END_REF] hearts subjected to long exposures to K-free solution. This hyperpolarisation may be attributed to a shift of the equilibrium potential for potassium ions in a negative direction.

In Purkinje cells, a similar effect was observed only during the first minutes in low [K + ] o . The large and sudden depolarisation observed at later times and the sudden repolarisation when returning to control [K + ] o were peculiar to Purkinje cells. Such de-and repolarisations have been studied in dog and sheep cardiac Purkinje fibres 4 5 26-29 (see also [START_REF] Elharrar | Voltage modulation of automaticity in cardiac Purkinje fibers[END_REF] for review) and were noted in fetal human ventricular myocardium. [START_REF] Gennser | Excitation and impulse conduction in the human fetal heart[END_REF] From the similarity of our results in human Purkinje cells to those from dog and sheep, we may infer that human Purkinje cells have a N-shaped steady-state membrane current-voltage (I(V)) relation with a region of negative slope-conductance. [START_REF] Gadsby | Two levels of potential in cardiac Purkinje fibers[END_REF] Lowering [K + ] o shifts the I(V) relation in the inward direction. [START_REF] Dudel | The potassium component of membrane current in Purkinje fibres[END_REF] (see fig 1 of [START_REF] Elharrar | Voltage modulation of automaticity in cardiac Purkinje fibers[END_REF] ) Thus the region of negative slope-conductance crosses and goes below the voltage axis. This accounts for a small and slow depolarisation followed by a large regenerative one [START_REF] Gadsby | Two levels of potential in cardiac Purkinje fibers[END_REF] (fig 5A). The reverse occurs on returning to control [K + ] o . 26 28 These effects have been attributed to a fall in membrane permeability to K + ions, [START_REF] Carmeliet | Chloride ions and the membrane potential of Purkinje fibres[END_REF] at least partly due to a decreased gK1. 34 Such large depolarisations were also found in response to lowered temperature 35 37 38 or to Na-K pump blockade by dihydroouabain or lithium ions. [START_REF] Isenberg | The effect of dihydroouabain and lithium ions on the outward current in cardiac Purkinje fibers. Evidence for electrogenicity of active transport[END_REF] Thus, [K + ] o -related changes in Na-K pump current [START_REF] Delèze | Possible reasons for drop of resting potential of mammalian heart preparations during hypothermia[END_REF] may also contribute. 26 36 In the present preparation, thallous ions could have been used to disclose the part played by Na-K pump blockade (as in the experiments of fig 4). This was not done, due to the limited time available for the experiment.

The fact that TTX hyperpolarises the membrane of Purkinje cells (fig 7 and 13) indicates that a steady sodium current flows, which could be partly responsible for the depolarisation once outward currents are sufficiently decreased. 10 26 Such depolarisations are not found in ventricular muscle fibres, 6 7 which may indicate that the Na-K pump current [START_REF] Carmeliet | Electrogenesis of the action potential and automaticity[END_REF] and/or the steady sodium current [START_REF] Gadsby | Two levels of potential in cardiac Purkinje fibers[END_REF] play a larger part in resting potential generation in Purkinje fibres.

LOW [K + ] o -INDUCED MODIFICATIONS OF MYOCARDIAL AND PURKINJE ACTION POTENTIALS

Ventricular action potentials were shortened at the level of the plateau and lengthened at the final repolarisation phase, during the first minutes in low [K + ] o (fig 2Bb). Thus low [K + ] o might increase the outward current at potentials. positive to about -20 mV and decrease it at potentials between the resting potential and -20 mV. [START_REF] Boyett | Cow ventricular muscle. I: the effects of the extracellular potassium concentration on the current-voltage relationship. II: evidence for a time-dependent outward current[END_REF] After longer periods, the inhibition of the Na-K pump [START_REF] Gadsby | Activation of electrogenic Na + /K + exchange by extracellular K + in canine cardiac Purkinje fibers[END_REF] and the resulting increase in [Na + ] i would cause the Na-Ca exchange to extrude less Ca 2+ . Increase in [Ca 2+ ] i can activate outward K + -currents 42 43 and induce premature repolarisation of the action potential. Indeed signs of Ca-overload were observed in the preparation: ie transient depolarisations and after-contractions as seen in fig 3. 9 21 These oscillations in membrane potential and mechanical tension were related to oscillations of [Ca 2+ ] i 44 45 and seen to pre-exist in the control state. [START_REF] Akselrod | Electromechanical noise in atrial muscle cells of the carp: a possible ionic feedback mechanism[END_REF] They are enhanced by Ca-overload. 44 45 A slow phase of late repolarisation appeared beyond 30 min in low [K + ] o (fig 2Bd). This phenomenon has already been observed in papillary muscles of man [START_REF] Prasad | Transmembrane potential and contraction of human heart in relation to potassium and ouabain[END_REF] and guinea-pig. 9 19 23 It occurred simultaneously with the twitch 7 9 19 23 (see also fig 3)and might be considered as a plateau caused by a transient inward current such as that which developed in Purkinje fibres poisoned with strophanthidin. [START_REF] Kass | Ionic basis of transient inward current induced by strophantidin in cardiac Purkinje fibres[END_REF] In Purkinje action potentials, a lengthening was observed at all levels of repolarisation (fig 5B).This may be accounted for by a depression of the electrogenic Na-K pump current. [START_REF] Gadsby | Activation of electrogenic Na + /K + exchange by extracellular K + in canine cardiac Purkinje fibers[END_REF] A direct depression of the membrane K + permeability [START_REF] Carmeliet | Chloride ions and the membrane potential of Purkinje fibres[END_REF] could also be involved.

Ventricular action potentials were shortened in low [K + ],while Purkinje ones were lengthened (figs 2B and 5B). A similar effect was found in simultaneously recorded ventricular and Purkinje action potentials of the perfused pig moderator band by Gettes and Surawicz. [START_REF] Gettes | Effects of low and high concentrations of potassium on the simultaneously recorded Purkinje and ventricular action potentials of the perfused pig moderator band[END_REF] In their study, the effective refractory periods were altered in the same way. This means that low [K + ],may increase the efficiency of the "gating mechanism" taking place in distal Purkinje fibres. 3 18

ROLE OF A TTX-SENSITIVE STEADY-STATE SODIUM CURRENT IN PURKINJE RESTING AND ACTION POTENTIAL GENERATION

The shortening of the action potential duration and the hyperpolarisation described above in low concentrations of TTX are similar to those described in dog cardiac Purkinje fibres. 12 13 Both effects may be attributed to a steady-state TTX-sensitive sodium current 10 that flows at potentials between -10 mV and the diastolic potential. 10 11 13 46 IMPLICATIONS FOR EXCITATION AND CONDUCTION The severe extracellular K + depletion seen in this study does not occur clinically but may be considered as a means of blocking the Na-K pump [START_REF] Gadsby | Activation of electrogenic Na + /K + exchange by extracellular K + in canine cardiac Purkinje fibers[END_REF] as in digitalis intoxication alone or combined with acute hypokalaemia. [START_REF] Cranefield | The conduction of the cardiac impulse[END_REF] Spontaneous activity may occur in Purkinje fibres at a low membrane potential, 6 47 which may account for some ventricular tachyarrhythmias observed in man with digitalis intoxication and/or acute hypokalaemia. [START_REF] Cranefield | The conduction of the cardiac impulse[END_REF] An increased difference between the durations of ventricular and Purkinje action potentials (as in low [K + ] o ) might improve the gating mechanism. However the earlier repolarisation of ventricular cells enables them to be prematurely re-excited. [START_REF] Cranefield | The conduction of the cardiac impulse[END_REF] Transient depolarisations in low [K + ] o were not described in the human ventricle. They were recently found in human Purkinje fibres in the presence of adrenaline or ouabain. [START_REF] Dangman | Electrophysiologic characteristics of human ventricular and Purkinje fibers[END_REF] When such transient depolarisations become suprathreshold, they trigger extrasystoles. This may account for the facilitator effect of hypokalaemia and digitalis on extrasystoles. 6 24 The steady sodium current takes part in the maintenance of the gating mechanism. [START_REF] Coraboeuf | Effect of tetrodotoxin on action potentials of the conducting system in the dog heart[END_REF] Agents that decrease that current (eg local anaesthetics) may in turn depress the gating mechanism.

I am grateful to Prof 0 Rougier, Dr C Ojeda, Dr J Simurda, Dr P Arnaud and Prof P Touboul for support and criticism and to Dr G Dureau for providing the myocardial samples. My thanks go to Mrs E Rameaux and Mrs J Barbéry for help in typing and artwork. This work was supported by grants from the "Faculté de Biologie Humaine, Université Claude Bernard" in 1979 and 1980, and from the "Institut National de la Santé et de la Recherche Médicale" (CRL no. 81-50-23).

FIG 1 :

 1 FIG 1: Oscilloscope records of ventricular and Purkinje action potentials in a strip of human papillary muscle. Note the triangular shape of the ventricular action potential and faster initial repolarisation, low plateau and longer duration of the Purkinje action potential.

  Within 10 min in low [K + ] o , the diastolic potential was progressively shifted in a negative direction (fig 2A). This hyperpolarisation developed slowly, which is accounted for by the slow time course of changes in the bath concentration. Beyond 10 min the diastolic potential shifted more slowly in a negative direction and reached -100 mV after 40 min in low [K + ] o , (fig 2A). During the first minutes in low [K + ] o , the action potential duration was shortened at the plateau level and transiently lengthened at the late repolarisation phase (fig 2Bb). At intermediate times (eg 10 min, fig 2Bc), the action potential duration was shortened to half that of control both at plateau and late repolarisation phases. At later times, a slow phase of final repolarisation appeared (fig 2Bd).

20

 20

FIG 3

 3 FIG 3 Ventricular myocardial membrane potential (lower trace) and tension (upper trace). The horizontal thick line shows the null potential. Left panel: at equilibrium in control solution. Right panel: after 70 min in low [K + ] o . Note change in tension scale from left to right.

  Fig 4A shows that, in another strip of human ventricle, the positive inotropy was reversed by thallous ions in the same way as after returning to control [K + ] o . Fig 4B shows that low [K + ] o -induced after-contractions in a human atrial trabecule were quickly reversed by thallous ions. Purkinje cells During the first minutes in low [K + ] o , Purkinje diastolic potential became more negative by about 10 mV (fig 5A and B) along a time course similar to that in ventricular cells. The action potential was lengthened in duration at all levels of repolarisation (fig 5B). After longer times in low [K + ] o (ie 6 min in fig 5), the diastolic potential was suddenly shifted by about 30 mV in a depolarising direction and remained at this level as long as [K + ] o was kept low. At this depolarised level of potential, slowly rising action potentials were elicited upon stimulation and were followed by a slow diastolic depolarisation (fig5C). After returning to the control solution (5.9 mmol.litre -1 K + ), a slow repolarisation of the diastolic potential began. The diastolic potential then suddenly shifted to a value 5 mV more negative than the initial control value. Afterwards the diastolic potential went slowly back to the control level (fig5A).

FIG 4 A

 4 FIG 4 A: Slow chart records of twitch amplitude in a strip of human ventricle. Upper trace: after 130 min in 0.5 mmol.litre -1 [K + ] o , 2 mmol.litre -1 thallous chloride were added (arrow). Lower trace: after a subsequent 30 min equilibration period in 0.5 mmol.litre -1 [K + ] o , control solution was switched on (arrow). Preparation issuing from a 4 months-old female with mitral stenosis. B: Human atrial preparation. Upper trace: after 10 min in 0.5 mmol.litre -1 [K + ] o , 2 mmol.litre -1 thallous chloride were added. Note fast return to control twitch amplitude. Lower trace: expanded time scale record showing that after-contractions were rapidly abolished by thallous ions. Preparation from a 7 years-old female with aortic stricture.In A and B driving rate: 20 stim.min -1 .

FIG 7

 7 FIG7 Effects of TTX on the electrical activity of a human cardiac Purkinje cell. Note the shortening of the action potential duration and negative shift of the diastolic potential. a: control: b: 10 -7 ,c: 6 x 10 -7 , d and e: 2 and 4 min in 5 x 10 -6 , f: 8 x 10 -6 mmol.litre -1 TTX.


Georges Christé
email: georges.christe@ujf-grenoble.fr

CHANNELS IN THE T-TUBULES OF CARDIOMYOCYTES AS SUGGESTED BY THE PARALLEL DECAY OF MEMBRANE CAPACITANCE, IK 1 AND IK ATP DURING CULTURE AND BY DELAYED IK 1 RESPONSE TO BARIUM

Keywords: T-tubule, Background inward rectifier K channels, ATP-sensitive K channels, Membrane compartmentation

Localization of K(+) channels in the tubules of cardiomyocytes as suggested by the parallel decay of membrane capacitance, IK(1) and IK(ATP) during culture and by delayed IK(1) response to barium.

Georges Christé

at each age) in a defined (DMEM) medium, the value of Cm and the magnitudes of the background inward rectifier current (IK 1 ) and of the 2,4-dinitrophenolinduced ATP-sensitive potassium current (IK ATP ). Cm, IK 1 and IK ATP all had decreased significantly by 51, 83 and 88%, respectively after 4 days of culture. Analysis using a single exponential decay function of time gave time constants of 10 respectively 2.6±0.2, 2.2±0.5 and 2.4±0.4 days, respectively. Linear regressions of IK 1 and IK ATP versus Cm had regression coefficients of 0.93 and 0.98, respectively. These observations are consistent with a strong link of the decay of IK 1 and IK ATP currents to that of Cm. Furthermore, the time course of changes in

Introduction

Potassium currents are important determinants of the electrical activity of cardiac ventricular muscle (for a review [START_REF] Carmeliet | Mechanisms and control of repolarization[END_REF]. The background inward rectifier current (IK 1 ) governs the resting potential and contributes to the late phase of action potential repolarization, while the ATP-sensitive potassium current (IK ATP ) current is involved during ischemic/hypoxic challenge to the myocardium. These currents also contribute to accumulation-depletion of potassium in extracellular spaces, either in the physiological state or in pathological states. In skeletal muscle, the localization of K + conductances in the T-tubule membrane leads to accumulation-depletion of K + ions that have important consequences on muscle function (Almers and Stirling, 1984, for review). In cardiac muscle, such consequences may not yet be evaluated, as the membrane distribution of K + conductances is not known. It was recently established that ventricular cardiomyocytes in primary culture lose their T-tubules and the associated membrane capacitance [START_REF] Lipp | Spatially non-uniform Ca 2+ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture[END_REF][START_REF] Mitcheson | Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture[END_REF]. We report here that cultured rabbit ventricular myocytes lose a large part of IK 1 and IK ATP currents in parallel with the reduction in cell membrane capacitance (Cm). Moreover, IK 1 changes upon rapid external application and washout of Ba ++ ions occur with a initial delay consistent with a model of diffusion-limited access to the IK 1 channels. These data provide arguments for the preferential localization of IK 1 and IK ATP conductances to the T-tubule membrane. The functional consequences of this localization are discussed.

Materials and Methods

Materials and methods, unless otherwise specified, are the same as previously described [START_REF] Christé | Propafenone blocks ATP-sensitive K channels in rabbit atrial and ventricular cardiomyocytes[END_REF].

Isolation of Cardiomyocytes

Under sterile conditions the heart of New-Zealand female rabbits weighting 1.5 to 1.7 kg were excised under deep anaesthesia (Xylazine 20 mg.kg -1 plus Ketamine 100 mg.kg -1 ) and placed at the bottom of a Langendorff perfusion system. Ventricular myocytes were isolated by a conventional enzymatic digestion method and cultured in DMEM medium. The medium was renewed every other day.

The investigation conforms with the Recommendation from the Declaration of Helsinki and the Guiding Principles in the Care and Use of Laboratory Animals.

The "external solution" for whole-cell current recording experiments contained (mM): NaCl 150; KCl 5.4; MgCl 2 1.2; CaCl 2 0.2; NaH 2 PO 4 0.905; HEPES 10; glucose 10; pH was adjusted to 7.4 with NaOH. The pipette solution contained (mM): K-aspartate 115; KCl 5; MgCl 2 7; K 2 -ATP 4; Na 2 -phosphocreatine 2; pyruvic acid 5; Na 2 -EGTA 5; HEPES 10, pH was adjusted to 7.2 with KOH.

DMEM medium was prepared from powder (GIBCO-Life Sciences Technologies) and supplemented with penicillin-G 60 mg/l, streptomycin 50 mg/l and NaHCO 3 25 mM. No serum or growth factor additives were used. All chemicals, including 2,4-Dinitrophenol (DNP) were of analytical grade and purchased from Sigma (Saint Louis, MO).

Whole cell current measurements

Cells were placed on a glass coverslip that constituted the bottom of a narrow well (3 x 15 mm) machined in a water-jacketed perspex block. The inflow of physiological solution was pre-warmed through a 10 cm water jacket. Its temperature was maintained at 35 ± 1 °C (as assessed using a 0.1 mm thermocouple) by circulating the water jackets with thermostated water. Whole cell currents were monitored with a List EPC5 patch-clamp amplifier.

Experiments were driven and recorded by a TL1 DA/AD interface and PClamp program suite (Axon Instruments). Cell capacitance (Cm) and series resistance (Rs) were evaluated as follows. Twenty current responses to ∆ ∆ ∆ ∆V=10 mV square depolarizing pulses of 12 ms duration were sampled at 25 KHz and averaged. A single exponential was fitted to the capacitance current decay, which provided estimates of the initial current I0, the time constant Tau and the steady state current Iss. Cm was derived using the relation:

Cm = Tau * (1/Rs + 1/Rm) with Rs = ∆ ∆ ∆ ∆V/I0 and Rm = (∆ ∆ ∆ ∆V/Iss -∆ ∆ ∆ ∆V/I0).
A voltage-clamp ramp protocol was used for recording membrane currents. From a holding potential of -80 mV, a square prepulse to -120 mV for 25 ms was followed by a depolarizing voltage ramp from -120 to +120 mV (2.5 mV.ms-1) and a reverse ramp from +120 to -120 mV. The whole cell current data in response to the repolarizing ramp were corrected off-line for estimated junction potentials, Rs-induced drop from command voltage and ramp-induced capacitance current as previously described [START_REF] Christé | Propafenone blocks ATP-sensitive K channels in rabbit atrial and ventricular cardiomyocytes[END_REF]).

The main artefact was due to real Rs values ranging 5 to 15 MΩ, which caused large departures of real voltage from the command voltage ramp. After corrections were applied to both the recordings in the absence of DNP (control) and in its presence, the magnitude of the maximal DNP-induced current representing IK ATP was measured at 0 mV after subtraction of control current.

To isolate the IK 1 current, a linear leakage current reversing at 0 mV was computed and subtracted, yielding the corrected current versus voltage relations as averaged and plotted in figure 1A. The leakage conductance was estimated for each cell using the current magnitude at the observed reversal voltage of the current suppressed by 100 µM BaCl 2 (i.e. -73 to -75 mV). The magnitude of the IK 1 current for each cell was then measured as the absolute difference in currents at -60 and -90 mV.

Data analysis and statistics

Membrane current data were processed under Clampfit and custom programs in Fortran-77 and MATLAB (The Math Works Inc.). Statistical analysis and graphs were done under ORIGIN 4.1 (Microcal Inc.). Data are given as mean ± S.E.. Comparison of means used unpaired t-test. The level of statistical significance was the 95% confidence level.

The time-dependent decay of cell capacitance or whole cell current magnitudes during culture was evaluated with an exponential function Y(t) of the form

Y(t) = (Y 0 -Y inf ) • exp(-t/τ) + Y inf (equation 1)
where Y 0 is the value of Y(t) at time 0, τ its time constant of decay and Y inf its steady-state value. This function was adjusted to the data using a least squares fitting routine. A linear regression routine was built from the Linfit algorithm of [START_REF] Bevington | Data reduction and error analysis for the physical sciences[END_REF] under MATLAB.

Fitting of equation 1 to data, as well as linear regression, took into account errors both in abscissa and ordinate data sets. Thus, the 95% confidence intervals that were computed for each estimated parameter value include the contribution of both errors from all data points. They are indicated near to parameter values in the text.

Theoretical model of Ba ++ diffusion and binding to the IK 1 channel

Changes in Ba ++ concentration in the T-tubules ([Ba] t ) upon changing the bath concentration ([Ba] e ) were represented by differential equation:

d[Ba] t /dt =([Ba] e -[Ba] t ) /τ τ τ τ T ) (equation 2)
where τ T was the time constant of exchange between bulk solution and T-tubule lumen. The fraction of available IK 1 channel (F t ) with binding of Ba ++ ions to a single external site was computed as:

dF t /dt = k 2 -(k 1 . [Ba] t + k 2 ) . F t (equation 3)
where k 1 and k 2 are microscopic binding and unbinding rate constants. Computation of the theoretical time course of unblocked IK 1 fraction was performed by 4th order Runge Kutta integration under MATLAB.

Results and Discussion

Features of IK 1 and IK ATP currents in freshly dissociated and in cultured cells

Figure 1A shows the average whole cell magnitude of the IK 1 current versus voltage in freshly dissociated myocytes (curve a). The current-voltage relation was similar to that reported by [START_REF] Giles | Comparison of potassium currents in rabbit atrial and ventricular cells[END_REF]. The IK 1 current was dramatically decreased after 3-5 days in culture conditions (curve b) and in a uniform way over the voltage range considered. A small outward current present at voltages positive to -20 mV was also decreased [START_REF] Mitcheson | Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture[END_REF]. The current induced by 100 µM DNP (Fig 1B) was fully blocked by 10 µM Glibenclamide, was weakly inward rectifying, reversed around -75 mV, thus it was termed IK ATP . Its magnitude was greatly decreased after more than 3 days in culture (fig 1B). For both currents, the shape of the current-voltage relations were poorly affected by time in culture.

Decay of membrane capacitance, IK 1 and IK ATP currents with time in culture

As shown in figure 2A, the whole cell capacitance progressively decreased with time in culture. From an initial value of 109±18 pF (n=10) on day 0, it decayed to 53±8 pF (n=9) after about 4 days. The decay of Cm was not significant at day 1, and became significant at later times. The loss of cell capacitance fitted with an exponential function (equation 1, solid line in Fig. 2A) proceeded with a time constant τ = 2.6±0.2 days (parameter estimate ± 95% confidence interval), and the estimated steady-state level (41.4±2.3 pF) was not reached after 5 days in culture. Such a decay of membrane capacitance in cultured ventricular myocytes has been mainly correlated with the disappearance of the T-tubules [START_REF] Lipp | Spatially non-uniform Ca 2+ signals induced by the reduction of transverse tubules in citrate-loaded guinea-pig ventricular myocytes in culture[END_REF][START_REF] Mitcheson | Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture[END_REF] while a minor part was due to decrease in cell size [START_REF] Mitcheson | Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture[END_REF]. The fraction of Cm lost as computed from the present data (0.62) agrees with the fraction of the cell membrane in the T-tubules as determined in ventricular myocytes by morphometric methods: 0.53 [START_REF] Amsellem | Transverse-axial tubular system in guinea pig ventricular cardiomyocyte: 3D reconstruction, quantification and its possible role in K + accumulation-depletion phenomenon in single cells[END_REF]. In about 4 days of culture, IK 1 (Fig. 2B) and IK ATP (Fig. 2C) currents decayed to 17% and 12% of their value at day 0, respectively. As for Cm, this decay became significant (p<0.05) from day 2 on. The IK 1 and IK ATP currents decreased slowly towards magnitudes not significantly different from 0 (Figs. 2B and2C). Their changes were fitted with a single exponential decay function (equation 1). The time constants of decay were estimated to 2.2±0.5 and 2.4±0.4 days respectively, they were not significantly different from that estimated to 2.6±0.2 days for Cm decay. Thus there was a close parallelism in the time course of disappearance of IK 1 and IK ATP with that of Cm.

Relation between current magnitudes and cell capacitance

The close parallelism of the changes in IK 1 and IK ATP with those of Cm suggests that these currents might be directly linked to the membrane pool that disappeared. To account for such a link, the hypothesis of a common determinant of their functional expression (genomic link) or of their maintenance in a functional state (e.g.: cytoskeletal rearrangement) might be invoked. Another possibility is that the IK 1 and IK ATP channels are both mainly localized in the Ttubule membrane. In support of the hypothesis of localization of the IK 1 current in cardiac T-tubules, the data of [START_REF] Mitcheson | Action potentials, ion channel currents and transverse tubule density in adult rabbit ventricular myocytes maintained for 6 days in cell culture[END_REF] show the IK 1 current to decay in parallel with Cm in left ventricular rabbit myocytes in culture, despite a 4 fold faster time course of decay in Cm (τ = 0.57 day) than reported here (2.6 days). This discrepancy might be related to the fact that we used uncoated culture dishes whereas Mitcheson et al. used laminin attachment, which was reported to accelerate the loss of T-tubules [START_REF] Delcarpio | Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells[END_REF]. A decrease of IK 1 during primary culture is also observed in ventricular myocytes from adults rats [START_REF] Wahler | Developmental increases in the inwardly rectifying potassium current of rat ventricular myocytes[END_REF][START_REF] Schackow | Electrophysiology of adult cat cardiac ventricular myocytes: changes during primary culture[END_REF] whereas T-tubule density is decreased [START_REF] Delcarpio | Ultrastructural morphometric analysis of cultured neonatal and adult rat ventricular cardiac muscle cells[END_REF]. Thus changes in IK 1 magnitude closely follow the amount of Ttubules in cardiomyocytes.

To our knowledge this is the first study to demonstrate a parallel decrease in IK ATP with Cm in cardiomyocytes during cell culture. The hypothesis of localization of IK ATP channels in the T-tubules is supported by the observation that, in skeletal muscle, the K ATP channel ligand P1075 preferentially binds to Ttubules [START_REF] Dickinson | Nucleotide regulation and characteristics of potassium channel opener binding to skeletal muscle membranes[END_REF].

In line with the hypothesis of T-tubule localization of IK 1 and IK ATP channels, the relation of cell current magnitudes of IK 1 and IK ATP to Cm values were analyzed by linear regression (see methods), which yielded regression coefficients of 0.93 and 0.98 respectively. The intercepts of the regression lines (Fig. 2D andE) with the Cm axis give an estimate of the capacitance of the membrane when tubular IK 1 or IK ATP currents have decayed to zero i.e.: 45 pF and 40 pF respectively. These values are similar to the steady-state value of Cm (41 pF), that corresponds to the average capacitance of the external membrane of cells. The slope of the regression lines, that give an estimate of the current density at the tubular membrane, is 37 pA/pF for IK 1 and 128 pA/pF for IK ATP .

Time course of external Ba effects on the IK 1 current

One consequence of the T-tubule localization of the IK 1 conductance would be a diffusion delay in the access of an external blocker to the IK 1 channels. This was tested using a local perfusion system (95% change in 50 ms) to apply 100 µM BaCl 2 in external solution to a freshly dissociated cell. The cell was placed in front of the 300 µm diameter outlet of a gravity-driven perfusion system (50 µl/min; [START_REF] Christé | Propafenone blocks ATP-sensitive K channels in rabbit atrial and ventricular cardiomyocytes[END_REF]) that allowed switching to a new solution with a pure delay of 4 seconds, the time for 95% change being estimated to 50 ms from the change in the liquid junction potential at the tip of a patch pipette upon change in KCl concentration from 5.2 to 0.5 mM. The time course of onset and washout of the effect of BaCl 2 was recorded at -120 mV (fig 3) and fitted by a simple model including diffusion in the T-tubules and binding of Ba ++ to the IK 1 channel (as described in the Materials and Methods).

In order to fit the data, parameters were initially set to values imposed by the actual data. The ratio k2/k1 (i.e.: the apparent Kd of the IK 1 channel for Ba ++ ions) was set to 1.3 µM at -120 mV, to account for the steady state fraction of IK1 remaining in the presence of 100 µM BaCl2. This is near to a Kd of 3.25 µM at -120 mV in guinea-pig ventricular myocytes [START_REF] Hirano | Barium-induced automatic activity in isolated ventricular myocytes from guinea-pig hearts[END_REF].

A detailed study of the mathematical system shows that the time course of onset of Ba effect mainly depends on k1 and τ T and that the time course of washout of the Ba effect is entirely defined by k2 and τ T . As a result, the system is constrained to a single set of final parameter values.

As may be readily noted from figure 3, the changes in the IK 1 current upon BaCl 2 application and wash followed a sigmoidal time course, which was reproduced when the time constant of diffusion (τ T ) was set to 300 ms. This value is in agreement with the time constants (250-270 ms) calculated by [START_REF] Levi | Diffusion of nickel, cadmium and calcium ions into the transverse (T) tubules of guinea-pig cardiac myocytes at 37°C (Abstract)[END_REF] for the access of divalent cation blockers to L-type Ca channels that are situated in the T-tubules (Gao et al. 1997). Thus the time course of BaCl 2 effects is consistent with the hypothesis that a large part of the IK 1 conductance is located in the T-tubule membrane.

As a whole, the results of this study support the hypothesis that IK 1 and IK ATP channels in ventricular cardiomyocytes are preferentially localized in T-tubules.

It is clear that a direct histological proof of the localization might be sought for, using appropriate specific antibodies against IK 1 and K ATP channels.

Physiological implications of such a localization of cardiac K + channels deserve consideration. It is known that the Na-K pump, Na-Ca exchanger as well as Na channels are present in the T-tubular membrane and that the T-tubule is the major site of E-C coupling in ventricular cells. In physiological conditions, accumulation of K + ions in the T-tubule lumen [START_REF] Freygang | The after-potential that follows trains of impulses in frog muscle fibers[END_REF] would tend to build up at high heart rates as a result of slow washout of K + ions flowing outward through the IK 1 channels. This might contribute to the supernormal excitability seen at high frequencies of stimulation, and also to shortening of action potential duration, as both a K + -induced increase in K + conductances and a depolarization of the T-tubule membrane are expected. Furthermore, through the interplay of luminal K + elevation, Na-K pump stimulation, local changes in intracellular Na + and in the Na-Ca exchange rate, the T-tubule may be the site of transient amplification phenomena taking part in the fine beat-to-beat tuning of E-C coupling.

In conditions of ischemia/hypoxia, further cumulative efflux of K + ions through K ATP channels [START_REF] Yasui | Contribution of potassium accumulation in narrow extracellular spaces to the genesis of nicorandil-induced large inward tail current in guinea-pig ventricular cells[END_REF][START_REF] Tourneur | SR47063, a potent channel opener, activates KATP and a time-dependent current likely due to potassium accumulation[END_REF]) may further depolarize the T-tubule membrane and cause triggered activities and eventually a loss of excitability. This may play a role in proarrhythmic as well as in protective effects (energy sparing due to impaired E-C coupling) known to occur in these pathological conditions. The functional implications of the possible localization of K + channels on the Ttubule function deserve further quantitative evaluation, both at the experimental and theoretical levels.

Legends to Figures

Figure 1 :

 1 Figure1: Superimposed whole cell current-voltage relations. Corrections were applied as described in Methods. After appropriate corrections, averages of whole cell current magnitudes were computed at each voltage during the descending ramp voltage protocol. A: average ± S.E. of data from 10 cells at day 0 (trace a) and 9 cells at day 3 through 5 (trace b) in control Tyrode. B: Average at day 0 (trace a, n = 6) and at days 3 through 5 (trace b, n = 4), of maximal DNP-induced outward current.

Figure 2 :

 2 Figure 2: Analysis of the decay of Cm, IK 1 and IK ATP with time in culture.The time dependent decay of Cm (A), and of the absolute magnitudes of the IK 1 current (B) and the IK ATP current (C) were fitted by a single exponential function with offset (continuous curves in A, B and C). Linear regression analysis was performed for IK 1 versus Cm (D) and for IK ATP versus Cm (E). The number of cells is given near each data point.

Figure 3 :

 3 Figure 3: Time course of block and unblock by external Ba ++ .The IK 1 current was continuously recorded at -120 mV. Plotted are the current values after after leak subtraction (corresponding to a 800 MΩ leakage membrane resistance) and normalization to maximal IK 1 (circles in A, scattered trace in B). A: response to the onset of Ba ++ ions perfusion that started at the up arrow, B:response to the subsequent wash of Ba ++ ions that started at the down arrow.Note the difference in time scales between the two panels. Continuous thick lines in both panels show the best adjustment along the model presented in Materials and Methods. Parameter values were: k 2 = 0.257 sec -1 , k 1 = 1.97 x 10 5 sec -1 .M -1 , τ T = 300 ms.

Acknowledgments

I am grateful to Dr. Yves TOURNEUR, Dr. Ian FINDLAY and Professor Jean VERDETTI for their critical reading of the manuscript and helpful suggestions.

Thanks go to Mrs Habiba TEBBAKH and Dr. Rémi FORRAT for contributing part of the experimental data.


Bin Li

Qianwen Guan

Zixia Fan

Mosi He

Speech production in noise by Chinese learners of English

Keywords: Lombard effect, speech production in noise, Chinese learners of English, lexical stress

This study examined effects of noise on speech production in first and second languages (L1 and L2) by examining correlates of segments and suprasegmentals produced by experienced Chinese learners of English. Vowels were significantly lengthened while consonants remained comparable under babble noise in both Chinese and English. Fundamental frequency was unchanged in Chinese, but raised in English in both quiet and noisy conditions. Phonetic realization of lexical stresses was partially maintained under noise to achieve contrast between strong and weak syllables. Our findings reveal Lombard effect in Chinese speakers' L1 and L2 speech production. Patterns of phonetic modification in Lombard speech appear language-specific, which may be attributed to L1 transfer or limited experience in L2 communication under adverse conditions.

INTRODUCTION

Speakers tend to involuntarily increase vocal efforts when speaking in adverse conditions such as under noise, which is referred to as the Lombard effects [START_REF] Lombard | Le signe de l'élévation de la voix [The sign of the rise of the voice[END_REF]. Lombard speech has been examined and found in a few languages, such as English [START_REF] Junqua | The Lombard reflex and its role on human listeners and automatic speech recognizers[END_REF], French [START_REF] Garnier | Speaking in noise: How does the Lombard effect improve acoustic contrasts between speech and ambient noise?[END_REF], Spanish [START_REF] Castellanos | An analysis of general acoustic-phonetic features for Spanish speech produced with the Lombard effect[END_REF], Japanese [START_REF] Takizawa | Lombard speech recognition by formant-frequency-shifted LPC cepstrum[END_REF], etc. Acoustically, Lombard effect is accompanied typically by an increase in vocal amplitude. However, previous studies found that modifications to other phonetic cues, such as duration and fundamental frequency (F0), were present in Lombard speech depending on phonetic contexts. For instance, acoustic adaptation in noisy conditions seemed to favour vowels rather than consonants. [START_REF] Junqua | The Lombard reflex and its role on human listeners and automatic speech recognizers[END_REF] found that vowels are more likely to be elongated while consonants slightly shortened. [START_REF] Garnier | Speaking in noise: How does the Lombard effect improve acoustic contrasts between speech and ambient noise?[END_REF] reported background noise induced greater changes in intensity and spectral energy of vowels than in those of consonants. In addition to fine-grained modification of segmentals, Arciuli and colleagues (2014) examined English lexical stresses in noise and found that English speakers did not increase vocal intensity equally across syllables, but altered degree of contrast between strong and weak syllables in Lombard speech. In the production of lexical stresses, both intensity and fundamental frequency showed greater differences between strong and weak syllables in noise than in quiet.

The phonetic modifications triggered by background noise are interpreted as attempts to facilitate speech communication in adverse conditions, where speakers employ measures available to enhance the intelligibility of speech in their native language. Much less is known, however, whether and how non-native speech shows such effect and modification, as second-language (L2) speakers who are limited by their linguistic experience may have more difficulty in identifying L2 speech targets in noisy conditions than native listeners (Li, Wayland, Zhang, 2012). For the production, [START_REF] Marcoux | Differences between native and non-native Lombard speech in terms of pitch range[END_REF] reported the non-native Lombard speech in terms of F0 range by examining native speakers of Dutch whose Lombard speech showed the influence of their native language. Previous studies warrant an investigation of the impact of noise on L2 speech production and remedial strategies if any by L2 learners.

STUDY DESIGN

This study aimed to investigate universality and embodiments of the Lombard effect by comparing native and non-native speech production under noise. To this end, we examined the Lombard effect in Chinese learners' English-as-an-L2 (ESL) speech production under noise, in terms of phonetic realizations of vowels, consonants, and lexical stresses. Much research has been devoted on ESL acquisition of suprasegmentals by learners whose native rhythmic mechanisms differ. Chinese learners are known to have difficulties in mastering English lexical stresses. In this study, we intended to examine the presence of Lombard effect in Chinese speakers' ESL production in noise with a reference to their L1. In other words, if L2 phonetic patterns by Chinese speakers shows any variation in noise from that in quiet, and any difference from their L1 production. As L2 learners may lack experience in dealing with degraded speech signals or selfmonitoring under adverse conditions, whichever patterns of interlanguage or L1 transfer they show in their L2 speech in a quiet condition may not be fully maintained under noise. L2 learners' phonetic performance, if different from that of native speakers in quiet, would remain different if not more distant in babble noise, because of the acoustic and verbal interference.

In order to elicit phonetic modification if any and for systematic comparison, we selected bisyllabic Chinese words varying in lexical tones and English words varying in the location of stress assignment. Altogether, we selected 18 Mandarin words, 30 Cantonese words, and 28 English words. The background noise in the noisy condition was synthesized from intensity-normalized babbles recorded in cafeteria and canteens. The babble noise was played out via loudspeakers at 76 dB while participants talked. Speech enhancement method using A Priori Signal-to-Noise Ratio [START_REF] Scalart | Speech enhancement based on a priori signal to noise estimation[END_REF] was adopted before acoustic analysis to filter out noise from speech signals. However, this method may suppress harmonics in speech signals during noise reduction, and thus cause undesirable degradation or normalization of intensity levels. Therefore, we did not include intensity in acoustic analysis, though it is an important cue to lexical stresses. The parameters to measure include duration of consonant, duration and peak F0 of vowel, the latter two of which were then fed to the formula below, where dk is duration or peak F0 of the kth syllable in a word, to calculate normalized pairwise-variability-index (nPVI) for stress realization. The nPVI reflects the degree and direction of contrastive stress across syllables in a word (Arciuli et al., 2014, Nolan and[START_REF] Nolan | The pairwise variability index and coexisting rhythms in language[END_REF]. A higher absolute nPVI indicates greater contrast between strong and weak syllables. nPVI = 100 × |(dk -dk+1) / ((dk + dk+1)/2)| We recruited three native speakers of Chinese who are experienced ESL users: two Mandarin speakers and one Cantonese speaker. They produced the word list in their L1 and then that in English, in both quiet and noisy conditions, totaling 300 trials. All recordings were then transcribed for phonetic analysis in Praat [START_REF] Boersma | Praat: Doing phonetics by computer (version 6.1.04)[END_REF].

For data analysis, we adopted linear mixed models in R (R Core Team, 2015) using the lme4 package [START_REF] Bates | Fitting linear mixed-effects models using lme4[END_REF]. A set of models was run with the tested acoustic parameters as dependent factors. The fixed factors were Condition (noise & quiet) and Language (L1 & L2) and the interaction between them. All models were specified for random intercept for Speaker and for Word. P-values were obtained by a likelihood-ratio-test in which the full model was compared to a model without the factor in question. If necessary, Tukey posthocs were conducted with the lsmeans package (Lenth and Hervao, 2008).

RESULTS AND DISCUSSION

Consonants

We measured duration of consonantal parts of all syllables. As presented in Table 1, consonants in noise were numerically shorter than their counterparts in L1 but sustained in L2.

Vowels

We measured duration and peak F0 of vowels. As listed in Table 2, vowels under babble noise were much longer than their counterparts in the quiet condition (χ2(2)= 8.33, p = 0.01) in both L1 and L2. Chinese speakers' F0 did not change in L1, but was raised in L2 in both quiet and noise. The spectral changes in F0 however, were not significant. Furthermore, for both consonants and vowels, language (L1 and L2) was found to interact with speaking conditions (vowels: χ2 (2)= 30, p < 0.0001; consonants: χ2 (2)= 8.49, p = 0.014). In other words, Chinese speakers adjusted to noisy conditions well in their native tongues in a similar way, but not as successfully in their L2, showing more individual variations in phonetic modifications.

Lexical stresses

To further compare the Lombard effect in L1 and L2, we examined ESL production of lexical stresses by calculating nPVI using duration and peak F0 of vowels in strong and weak syllables. There were both trochaic and iambic words on the English word list, and thus SW and WS patterns.

In quiet condition, durational contrast in SW words is significantly smaller than that in WS ones, and the pattern is maintained under noise. The variation pattern between trochaic and iambic words is consistent with previous findings [START_REF] Li | Phonetic cues of lexical stress in Chinese learner English[END_REF]. But the mean range in SW words slightly shrinks under noise. Moreover, nPVI of vowel duration showed no general difference for either SW or WS syllables in quiet or noisy conditions, as shown in Figure 1.

General Discussion

Results from acoustic analysis reveal patterns of modification in Lombard speech by Chinese speakers. In general, consonants were not lengthened as much as vowels were in noise. The temporal increment in vowel production under noise is significant in both L1 and L2. The nPVI reveals that durational contrast of syllables in trochaic words was weakened, suggesting durational lengthening may have been applied to all syllables. Chinese speakers' F0 in L1 was the same in quiet and noise, which is expected in tonal languages. Interestingly, their peak F0 was raised when producing English in quiet and further in the noise condition.

The tendency of modification in F0 and duration echoes previous findings that Chinese ESL learners may use a falling tone to signal a stress or apply a high tone overall while lengthening a stressed syllable [START_REF] Lai | Acoustic Realization and Perception of English Lexical Stress by Mandarin Learners[END_REF][START_REF] Zhang | Acoustic characteristics of English lexical stress produced by native Mandarin speakers[END_REF]. In our study, Chinese ESL users adopted both temporal and spectral methods in achieving the phonetic contrast between strong and weak syllables, which were sustained under noise. The slight weakening of contrast in terms of vowel duration in SW words prompts certain impact of noise on temporal adjustment. Noise may have distracted the speakers' attention to phonetic details. They engaged much effort in filtering out interference from noise and that from linguistic cues in babbles. Cognitive load in maintaining clear speech may have been too demanding for nonnative users to allot sufficient attention to phonetic accuracy at a higher-level in the target language. There is, however, another interpretation of the changes to phonetic contrast under noise. Chinese speakers could have realized the impact of noise on their speech and thus adopted the general strategy of cue enhancement, including F0 increment and vowel lengthening in L2 and in noise. Compensation to signal degradation may override that to phonetic cues, therefore their modification was implemented in an overall manner on all syllables, softening the contrast between strong and weak syllables rather than maintaining or lest enhancing it.

SUMMARY

This study investigated effects of noise on L2 production of segments and suprasegmentals by comparing phonetic correlates of consonants, vowels and lexical stresses by Chinese ESL users in quiet and in noise. Results confirmed Lombard effects in Chinese speakers' L1 and L2 production. Modification in amount and strength is observed more in vowels than consonants in noise, which is consistent with [START_REF] Junqua | The Lombard reflex and its role on human listeners and automatic speech recognizers[END_REF] that reported greater Lombard effect in native speakers' production of vowels than in that of consonants. Moreover, in contrast to some previous studies [START_REF] Junqua | The Lombard reflex and its role on human listeners and automatic speech recognizers[END_REF][START_REF] Garnier | Influence of sound immersion and communicative interaction on the Lombard effect[END_REF], fundamental frequency (F0) was maintained under the noise condition in L1 Chinese but slightly raised in L2 English. This finding confirms L1 influence in ESL speech production and language-specific strategies in phonetic modifications in Chinese and English. ESL data also suggest a potential trade-off relation between responses to general speech degradation and modification to fine phonetic details. But this hypothesis should be examined using wellcontrolled tonal contexts since Chinese (both Cantonese and Mandarin) is a tone language. Future research could test hypotheses of our study using more acoustic parameters and recruiting a larger pool of speakers.

ACKNOWLEDGEMENT

This study is supported by EDB(LE)/P&R/EL/175/12 from SCOLAR of Hong Kong and by Project 6391207 from the UGC T&L Scheme.

BIBLIOGRAPHY

Figure 1 :

 1 Figure 1: nPVI for vowel duration (upper panel), and peak F0 (lower panel) of English words produced by Chinese speakers in noise and in quiet.

Table 1 .

 1 Mean duration of consonants. (Values in brackets are standard deviation)

		Language	Condition	Mean (SD) P value
	Duration	L1	Quiet	96.1 (47.3)	0.24
	(ms)		Noise	90.2 (47.8)
		L2	Quiet	113.2(51.4)	0.92
			Noise	113.2 (53)
	P-values were determined by least-squares means comparison
	using the lsmeans package in R.

Table 2 .

 2 Mean duration and peak F0 of vowels. (Values in brackets are standard deviation) P-values were determined by least-squares means comparison using the lsmeans package in R.

		Language Condition Mean (SD)	P value
	Duration	L1	Quiet	158.4(61.4)	0.04
	(ms)		Noise	167.6(62.5)
		L2	Quiet	123.3(64.2)	0.03
			Noise	133.4(65.6)