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Cardiovascular Research 2003 57(4):1025-1034; doi:10.1016/S0008-6363(02)00645-4
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Copyright © 2003, European Society of Cardiology

Is there a transient rise in sub-sarcolemmal Na and activation of Na/K pump current following activation of INa in ventricular myocardium?

Benjamin d.Z Silvermana, Alice Warleya, Jonathan I.A Millera, Andrew F Jamesb and Michael J Shattocka,*

aCentre for Cardiovascular Biology and Medicine, The Rayne Institute, St. Thomas’ Hospital, King's College London, London SE1 7EH, UK
bDepartment of Physiology, School of Medical Sciences, University of Bristol, University Walk, Bristol BS8 1TD, UK

* Corresponding author. Tel.: +44-20-7928-9292x3376; fax: +44-20-7928-0658. michael.shattock{at}kcl.ac.uk

Received 7 January 2002; accepted 21 August 2002


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: The primary aim of this study was to investigate whether activation of Na influx via voltage-gated Na channels can elevate sub-sarcolemmal (‘fuzzy-space’) [Na] and transiently activate Na/K pump current (Ip). Methods and results: Initially, Na/K pump activity was characterised in whole-cell voltage-clamped single guinea-pig ventricular myocytes. Ip was activated by intracellular Na with a Km of 15.5 mM and a Hill coefficient of 1.7. Extracellular K activated Ip with a Km of 1.6 mM. In these experiments, a finite ouabain-sensitive Ip was measured when the pipette [Na] was zero. This suggests that there is an accumulation of Na in a sub-sarcolemmal space that is not in equilibrium with the bulk cytosol (which is assumed to be efficiently dialysed by the low-resistance patch-pipettes used). Such a sub-sarcolemmal Na gradient was observed in separate experiments in intact rabbit papillary muscles using electron probe X-ray microanalysis. In these studies, a fuzzy-space of limited Na diffusion was observed 100–200 nm below the sarcolemmal membrane. This sub-sarcolemmal Na gradient was similar whether muscles were frozen at peak-systole or end-diastole suggesting that the fuzzy-space Na does not change over the course of the contractile cycle. This was further investigated in isolated guinea pig myocytes where evidence for a transient activation of Ip was sought immediately after the activation of voltage-gated Na channels. A single clamp step from –80 to 0 mV activated Na influx but, in the 10–2000 ms immediately following the initial Na influx no evidence for a transient activation of Ip was observed. Similarly, no activation of Ip could be detected immediately following a train of 20 rapid (5-Hz) pulses designed to maximise Na influx. Conclusions: These studies provide evidence for the existence of a maintained sub-sarcolemmal elevation of [Na] in ventricular myocardium; however, this fuzzy-space [Na] did not change immediately after the activation of Na influx via voltage-gated Na channels or throughout the contractile cycle.

KEYWORDS Electron microscopy; Intra/extracellular ions; Ion channels; Ion pumps; Myocytes; Na/K-pump


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
The Na/K pump is found at the plasma membrane of all animal cells [1] and provides the major route for cellular Na extrusion [2,3]. The energy derived from the hydrolysis of one ATP molecule is used by the Na/K pump to extrude three Na ions in exchange for two K ions, and this stoichiometry is maintained over a wide range of extracellular potassium concentrations ([K]o), intracellular sodium concentrations ([Na]i) and membrane potentials [4–7]. The net flux of positive charge from the cell via the Na/K pump is associated with an outward membrane current (Ip) [8,9].

A principal modulator of Na/K pump current is the intracellular Na concentration ([Na]i) [10–12]. In cardiac muscle, during periods of rapid pacing, bulk cytoplasmic [Na]i rises [13] and this activates the Na/K pump [14]. The outward pump current stimulated by rapid pacing in turn hyperpolarizes the resting membrane potential and contributes to shortening of the action potential (for review, see Gadsby [14]). While such observations have been made in a number of preparations and by many authors, they require only a rise in bulk intracellular [Na]i. However, there is evidence from a number of approaches that intracellular Na may be spatially heterogeneous (see review by Carmeliet [15]) and may also change dynamically during the contractile cycle [16–20].

Within the time-course of the action potential there is evidence that Na may be transiently elevated in a sub-sarcolemmal ‘fuzzy-space’ following a voltage-gated Na current (INa) and this in turn activates reverse-mode Na/Ca exchanger activity to initiate Ca influx [16–20]. Such a dynamic and transient rise in sub-sarcolemmal Na ([Na]ss) should, in principle, also transiently activate the Na/K pump current. As with the activation of Na/Ca exchange, this transient activation of the Na/K pump would occur immediately after the completion of INa and the termination of the upstroke of the action potential. Thus, theoretically, a transient outward Na/K pump current, activated by a brief rise in [Na]ss, could contribute both to early repolarisation and may also influence action potential duration. Su et al. [21] have recently suggested that the ability of Na influx following INa to influence reverse-mode Na/Ca exchange and the Ca transient is modulated by the Na/K pump. This would imply that Na influx via voltage-gated Na channels may at least transiently activate the Na/K pump. To date a transient activation of the Na/K pump current following Na influx has not been investigated.

The principal aims of this study were (i) to characterise Na/K pump currents recorded in guinea pig myocytes, (ii) to use this model to assess whether Na channel activation leads to a transient increase in the Na/K pump current (Ip), and (iii) to use electron probe X-ray microanalysis (EPXMA) to investigate directly sub-sarcolemmal [Na] gradients in isolated rabbit papillary muscle—a model in which tissue can be snap frozen with high temporal precision either in systole or diastole.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Animals
All studies involved the use of either ventricular myocytes or papillary muscles isolated from guinea pig or rabbit hearts, respectively. All animals were maintained humanely in compliance with the Principles of Laboratory Animal Care formulated by the National Society for Medical Research and Guide for Care and Use of Laboratory Animals prepared by the National Academy of Sciences and published by the National Institutes of Health (NIH Publication No. 85-23, revised 1996).

2.2 Cell isolation
Ventricular myocytes were obtained using a standard enzymatic dissociation procedure from male Dunkin–Hartley guinea-pigs following cervical dislocation [22,23].

2.3 Recording arrangements
Single isolated guinea-pig ventricular myocytes were whole-cell voltage clamped as originally described by Hamill et al. [24]. Patch electrodes were made from borosilicate glass capillaries (Clark Electromedical Instruments, Reading, UK) and fire-polished (DMG Universal Puller, Zeitz-Instrumente Ventreibs GmBH, Germany). The electrodes had a resistance of 1–2 M{Omega} when filled with the standard pipette solution. The patch membrane was ruptured by a brief period of suction once a tight seal (R5 G{Omega}) had been established. Current signals were recorded using an Axopatch 1-C single electrode voltage-clamp amplifier (Axon Instruments, USA) via a CV-3 headstage (Axon Instruments) and controlled by a microcomputer running pClamp software (v.6, Axon Instruments).

2.4 Solutions
Unless otherwise stated all work was performed at 35 °C. Solutions were made up using purified water (Elgastat UHP, Elga Ltd., UK) with Analar Grade chemicals (BDH, UK). For the Na/K pump current measurements the standard pipette solution contained (in mM): NaCl 10, aspartic acid 100, CsOH 100, CsCl 20, TEA chloride 20, HEPES 10, EGTA 5, MgATP 5, creatine phosphate (Tris) 5, MgCl2 2, pH 7.16. In those pipette solutions in which NaCl was not 10 mM, equimolar concentrations of CsCl were used to replace the NaCl, or NaOH replaced equimolar concentrations of CsOH. The standard extracellular superfusate contained (in mM): NaCl 140, glucose 10, HEPES 5, KCl 5, NiCl2 2, MgCl2 1, BaCl2 1, pH 7.36. Changes in [K]o were achieved by altering the amount of KCl in the solution. In order to inhibit the Na/K pump, 100 µM ouabain (Sigma Chemical Co., St. Louis, MO, USA) was added to the extracellular superfusate or all KCl was excluded.

For the investigation of Na channel Erev, experiments were performed at room temperature (22–24 °C) and the pipette solution contained (in mM): NMDG 130 (Sigma), NaCl 10, TEA·Cl 18.2 (Sigma), aspartate 100 (Sigma), MgCl2 0.9, BAPTA 5 (Sigma), HEPES 10, MgATP 4, pH 7.2. The extracellular solution contained (in mM): TEA·Cl 125 (Sigma), NaCl 10, KCl 5, MgCl2 1.2, BaCl2 2, NiCl2 2, glucose 11, HEPES 10 and nifedipine (10 µM—from a 10 mM stock in ethanol), pH 7.35 with TEA·OH (Sigma).

2.5 Voltage-clamp protocol
Initial characterisation of the Na/K pump current (Ip) used a voltage ramp protocol. The ramp protocol initially increased Vm to +40 mV (at 65 mV/s), before decreasing it to –90 mV at –65 mV/s. This descending phase of the protocol was used to derive a complete I–V relationship for Ip. This ramp protocol was validated by comparison with results obtained at the end of 200 ms square-step depolarisations to between –80 and +40 mV. The relationship between Ip and [K]o (0–10 mM) was characterised with a pipette [Na] of 50 mM and the relationship with [Na]pip (0–50 mM) was characterised with 5 mM [K]o. Best fit relationships were obtained using Prism 2.0 software (GraphPad Power Inc., USA).

In order to study the influence of INa on subsequent Ip, the ouabain-sensitive current during a square-step depolarisation from a holding potential of –80 mV to zero was used. Depolarisation to zero was used as it is a potential at which Na/K pump current is near maximal and INa is still large (pilot experiments demonstrated that in normal test solution maximal Na channel current (INa) occurred on depolarisation to around –40 mV (not shown)). The step was maintained for a duration of either 40 or 2000 ms. Further studies used a train of nineteen 40 ms depolarisations at a frequency of 5 Hz followed by either a 40- or a 2000-ms depolarisation. These recordings were performed with a pipette [Na] of 10 mM, and [K]o of 5 mM. Finally, recordings were made with pipette [Na] of zero and also with step depolarisations from –80 to +40 mV.

In order to assess the effect of repeated Na channel activation on Erev for the Na inward current itself, a protocol of 50 ms voltage steps was performed on myocytes whole-cell voltage-clamped from a holding potential of –80 mV. The membrane potential was stepped to –30, –20, –10, 0, +10, +20, and +30 at ~5 s intervals, returning to –80 mV each time. The same voltage steps were then performed individually 180 ms after conditioning trains of 20 ms depolarisations to –30 mV at a frequency of 5 Hz. Peak Na channel currents were used to assess Erev. The pipette and bath solutions were designed to provide symmetrical Na concentrations and were as described above. In studies in which the Na/K pump was inhibited, K-free solutions were made by omitting KCl, and TEA·Cl was increased to 130 mM to maintain ionic strength. The switch to the K-free solution was achieved immediately prior to initiating the train of conditioning pulses using a PC-controlled rapid solution changer [25].

2.6 The isolated vascularly-perfused rabbit papillary muscle
The preparation is a modification of that previously described by Kléber et al. [26]. Briefly, hearts were excised from anaesthetised (75 mg/kg Na pentobarbital i.v.) and heparinised (1000 IU i.v.) male New Zealand White rabbits (2.5 kg). Following brief retrograde perfusion through the aorta, the atria, large blood vessels and left ventricular wall were removed. The septal artery was perfused with high K solution at a constant pressure of 40 cmH2O, and the right ventricular wall was removed along with any unperfused segments of the septum. The high K solution contained (in mM): NaCl 110, KCl 16, NaHCO3 25, MgSO4 1, CaCl2 1.8. The preparation was then placed in a perspex chamber at 35 °C with an atmosphere of moist 95% O2, 5% CO2. The preparation was perfused with modified KH buffer and stimulated at 2 Hz. The modified buffer contained (in mM): NaCl 118, KCl 3.8, KH2PO4 1.2, NaHCO3 25, MgSO4 1, glucose 10, CaCl2 1.8. Freeze-clamping was performed using a purpose-built cryoclamp and timing circuit (driven by the muscle stimulator) which allowed the muscle to be rapidly frozen at a precise and defined moment in the cardiac cycle [27].

2.7 Electron probe X-ray microanalysis
EPXMA was performed on sections from the freeze-clamped papillary muscle to measure subsarcolemmal [Na] at peak systole (80 ms after the upstroke of the action potential) and at the end of diastole. Sections, 200 nm thick, were cut from the muscles at –120 °C using a Reichert FC4 cryo-ultramicrotome. The sections were transferred onto Pioloform-covered hexagonal 100 mesh nickel grids. The grids were placed onto a pre-cooled brass carrier and freeze-dried overnight under controlled conditions using an Emitech K775 freeze drier (Emitech, Ashford, UK). After reaching room temperature the grids were coated with a thin layer of carbon while still under vacuum.

The sections were analysed in a scanning transmission electron microscope in spot mode at ambient temperature using a Zeiss EM 10 electron microscope fitted with a Link AN 10,000 microanalysis system (Oxford Instruments, High Wycombe, UK). The static spot (size 40 nm) was placed at measured distances from the sarcolemma and analyses were carried out 100 s live-time at 80 kV accelerating voltage and 1 nA beam current. Spectra were processed to remove the background and obtain net peak integrals using the supplied filtered least-squares fitting routine. Quantification was achieved by use of the continuum-normalisation procedure with reference to standards composed of gelatin containing known amounts of inorganic salts [28].

2.8 Statistical analyses
Data are expressed as a mean±S.E.M. and statistical significance was assessed by either two-tailed t-tests or analysis of variance (ANOVA) followed by Dunnett's corrected t-test; P<0.05 was considered significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Characterisation of Ip
Fig. 1 shows the K-dependent current measured using the ramp (–65 mV/s) voltage protocol compared to measurements made using a square-step protocol after 150 ms of equilibration. The two sets of data overlie each other and hence this ramp protocol provides a rapid and easy way to generate a complete quasi-steady state I–V relationship. Fig. 2 shows the relationship between Ip and [K]o measured at 0 mV during ramp protocols. From the fitted curve (see figure legend for details), the Km and Imax for current activation were estimated to be 1.6 mM and 2.3 pA/pF, respectively. Fig. 3 shows the relationship between Ip and [Na]pip. A significant current of 0.131±0.039 pA/pF was measured with zero [Na]pip. From the fitted curve (see figure legend for details), the Km and Imax for current activation were estimated to be 15.5 mM and 2.1 pA/pF, respectively. The relationships between [K]o and [Na]i and Ip measured at 0 mV shown in Figs. 2 and 3Go were similar at all potentials between –60 and +40 mV (not shown).


Figure 1
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  		Fig. 1 The K-dependent Na/K pump current measured using a step protocol and with a ramp protocol. The open circles represent the average data during the last 50 ms of a 200-ms duration at the potential. The continuous line represents the average data from a ramp protocol during which Vm was decreased from +40 to –90 mV at –65 mV/s (n=10, from three cell isolations).

 

Figure 2
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  		Fig. 2 Relationship between extracellular [K] and ouabain-sensitive Na/K pump current. The current sensitive to 100 µM ouabain was measured at 0 mV using the voltage-ramp protocol, with [Na]pip=50 mM (means±S.E.M.). The relationship was fitted by a hyperbolic function of the form: Ip=Imax[K]o/[K]o+Km). No values were fixed, although implicit in this equation is the assumption that at zero [K]o the value of the ouabain-sensitive Ip is zero [10]. Since the best fit is found by logarithmic calculation, the point at zero [K]o was excluded from the analysis. Best fit analysis indicated a Km of 1.6 mM and Imax of 2.3 pA/pF. The numbers adjacent to the data points indicate the number of cells studied.

 

Figure 3
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  		Fig. 3 Relationship between pipette [Na] and ouabain-sensitive Na/K pump current. The current sensitive to 100 µM ouabain was measured at 0 mV using the voltage-ramp protocol, with [K]o=5 mM (means±S.E.M.). The relationship between [Na]i and Ip was fitted by a sigmoidal function of the form: Ip=Imin[(Imax–Imin)/(1+(Km/[Na]i)n)]. In this equation, n represents the Hill coefficient. Since the best fit is found by logarithmic calculation, the point at zero [Na]i was excluded from the analysis. However, the Imin value was fixed at 0.131 (the measured Ip when pipette [Na] was zero). The best fit indicated a Km of 15.5 mM, n of 1.7 and Imax of 2.1 pA/pF. The numbers adjacent to the data points indicate the number of cells studied.

 
3.2 Is there a transient Na/K pump activation following INa?
Fig. 4 shows typical whole-cell responses to a depolarisation from –80 mV to zero, before and after Ip block with ouabain. The typical result achieved without ouabain present (Fig. 4a) showed a capacitance spike (upgoing) followed within 2 ms by the inward INa which was completely inactivated within 10 ms after the depolarising step, leaving a positive holding current of about 100 pA. In the presence of ouabain (Fig. 4b), this holding whole-cell current was reduced to close to zero after INa inactivation. Fig. 4c shows the subtracted Na/K pump current (Ip) measured during the 40 ms clamp step. Fig. 5a shows the mean results from four cells. There was no evidence of a transient increase in Ip following the depolarising step (P0.05, paired t-test). To avoid potentially missing a transient increase in Ip over a longer duration, a depolarisation of 2000 ms was used. Fig. 5b shows that there was no evidence of a transient activation over this longer duration (P0.05). Fig. 6 shows Ip during the last in a train of repeated depolarisations at 5 Hz. Again, there was no transient increase in Ip following inactivation of the Na channel current (P0.05). In order to exclude the possibility that, in these experiments, Ip was saturated, further experiments were performed using a pipette [Na] of zero or using a depolarising step to +40 mV to limit Na accumulation during INa. Neither protocol revealed any evidence for a transient activation of Ip following INa (data not shown, P0.05). Further evidence against the saturation of Ip in the experiments in Figs. 4–6GoGo is that the ouabain-sensitive pump current was typically around 0.75 pA/pF—values that represent only about 30–40% of that maximally available (see Figs. 2 and 3Go).


Figure 4
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  		Fig. 4 A typical whole-cell response to a step depolarisation from –80 mV to zero for 40 ms. Both the initial capacitance spike and the ensuing Na channel current saturated the amplifier, as did the capacitance spike seen when Vm was stepped back to –80 mV. (a) Without ouabain present. (b) With 100 µM ouabain in the test solution. (c) Ouabain-sensitive Na/K pump current (Ip) obtained by subtracting the trace shown in (b) from that in (a).

 

Figure 5
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  		Fig. 5 Mean Ip following a step depolarisation from –80 mV to zero for a duration of (a) 40 ms, and (b) 2000 ms. Values represent the current during 10 ms bins centred at 15, 25, and 35 ms (means±S.E.M., n=4, from four animals) and during 100-ms bins centred at 250, 500 ms, etc. (means±S.E.M., n=4, from four cell isolations). Current values are normalised to cell capacitance.

 

Figure 6
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  		Fig. 6 Mean Ip following a step depolarisation from –80 mV to zero after a conditioning train of depolarisations. The conditioning train consisted of nineteen depolarisations at 5 Hz. (a) Ip during a 40-ms duration (n=4, from four animals). (b) Ip during 2000 ms duration (n=4, from two cell isolations). Current values are normalised to cell capacitance (means±S.E.M.).

 
3.3 Effect of INa on Erev for the Na channel
Fig. 7a shows the whole-cell response to depolarisations from –80 to –20 mV, zero and +20 mV, indicating activation and inactivation of INa. These measurements were made in symmetrical Na solutions, designed to assess INa (see Section 2). The upper traces in Fig. 7a show currents obtained during a single voltage step after a period of rest (first pulse) and currents recorded after twenty rapid (5 Hz) depolarisations to –30 mV (to repeatedly activate INa). Under control conditions, the current–voltage (I–V) relationship (Fig. 7b) passes close to the origin, as expected of INa when symmetrical [Na] is used in the internal and external solutions and the reversal potential was unaffected by repeated activation (Fig. 7b, closed circles). This suggests that, under these conditions of reduced Na influx, there is no accumulation of Na in the subsarcolemmal space (i.e. Na that comes in on INa is extruded from the cell in the period between successive pulses) and that a rise in bulk Na is effectively buffered by pipette dialysis. However, when Na extrusion is prevented by rapidly switching to a K-free solution immediately prior to the conditioning train (Fig. 7a, lower traces, and Fig. 7c), Na accumulates in the cell such that, after 20 conditioning pulses, the I–V relationship constructed on the 21st pulse shows a shift in the reversal potential for INa by approximately –12 mV. This would equate to a rise in intracellular Na sensed by the Na channel from 10 mM to around 16 mM.


Figure 7
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  		Fig. 7 The effect of a rapid train of 20 depolarisations on the peak Na channel current. (a) Typical whole cell responses to depolarisations from –80 mV to pipette potentials of –20 mV, zero and +20 mV before (left-hand panels), and after (right-hand panels), trains of 20 conditioning depolarisations (to –30 mV at 5 Hz). Experiments were performed either in control solution containing 5 mM K (upper panels) or after changing to K-free solution immediately after the initial pulse to inhibit Na/K pump activity (lower panels). (b) Mean INa at different depolarisations before (open circles) and after (filled circles) a train of conditioning depolarisations (means±S.E.M., n=4) in control conditions. (c) Mean INa at different depolarisations before (open squares) and after (filled squares) a train of conditioning depolarisations (means±S.E.M., n=4) following Na/K pump inhibition by removal of external K. Current–voltage relations in (b) and (c) have been corrected for the voltage-drop across the series resistance (–16.8±3.3 mV).

 
3.4 EPXMA measurements of [Na]ss
The data thus far demonstrate that there is no transient activation of Ip nor shift in the reversal potential of INa following INa activation when the Na/K pump remains active. This suggests that there is no transient accumulation of Na in a sub-sarcolemmal fuzzy-space early after electrical excitation in isolated guinea pig myocytes. In order to investigate this further, we have used electron-probe X-ray microanalysis to measure directly [Na]ss at two time points in systole and diastole. This necessitated the use of a different myocardial preparation (the isolated vascularly-perfused rabbit papillary muscle) to enable snap-freezing at defined time-points within systole and diastole with a temporal precision of ±1 ms [27]. While we accept that this introduces the complication of comparing between species and preparations, we were constrained to use our established rabbit model because of the prohibitive complexities of setting up rapid cryofixation in isolated myocytes. Fig. 8a shows an electron micrograph of a typical section of a papillary muscle frozen at end-diastole. [Na] was typically measured on a transect approximately half-way between the Z- and the M-lines perpendicular to the sarcolemma as indicated by the dotted line. Fig. 8b shows a typical [Na] profile measured along this transect showing a subsarcolemmal accumulation of Na that declines towards the centre of the cell with an approximate exponentially space-constant of, in this case, 41 nm.


Figure 8
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  		Fig. 8 (a) Electron-micrograph showing a typical section of a rabbit papillary muscle frozen at end-diastole. The dotted line indicates the position of the transect taken for EPXMA of [Na]. (b). Typical EPXMA values for [Na] are shown as a function of distance from the sarcolemma. The line represents a single exponential fit that, in this case, has a space-constant of 41 nm and is asymptotal to 94 mmol/kg dry weight.

 
Fig. 9 shows the mean [Na] measured at different distances under the membrane of rabbit papillary muscle cells, measured with EPXMA in muscles frozen at end-diastole and at peak systole (80 ms after the stimulus). The values are normalised to the value at the cell centre. The mean absolute values for [Na] at the cell centre were not significantly different between diastole and systole and were, respectively, 75±29 (n=7) and 84±16 (n=4) mmol/kg dry weight. Close to the sarcolemma, [Na] was similarly elevated in muscles frozen at peak systole or at end-diastole. Absolute values for [Na] measured 50 nm below the sarcolemmal membrane were 122±40 (n=7) and 198±53 (n=4) mmol/kg dry weight in diastole and systole, respectively. The mean values of [Na] shown in Fig. 9 were fitted with a single exponential curve as described in the figure legend and the derived space-constants for diastole and systole were estimated to be 52 and 123 nm, respectively. While there is some suggestion that the sub-sarcolemmal elevation of [Na] is greater and extends further into the cell in systole than it does in diastole (see Fig. 9), there were no significant differences between the curves describing the two data sets.


Figure 9
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  		Fig. 9 Mean sub-sarcolemmal [Na] distribution during systole and diastole measured using EPXMA. Aerobically perfused rabbit papillary muscles were frozen at peak systole (80 ms after the stimulus; open circles) or at the end of diastole (closed circles) during maintained steady-state stimulation at 2 Hz. [Na] was measured as described in the text and values are normalised to [Na] at the centre of the cell. Each data set is fitted with a single exponential curve according to the equation Y=Ymax*exp(–(0.69/{lambda})*x)+100, where {lambda} is the space-constant. Ymax and {lambda} were estimated to be 205% and 52 nm for diastole and 191% and 123 nm for systole, respectively. Values represent data sets derived from either four (systole) or seven (diastole) muscles (mean±S.E.M.) for each group where each data set represent the means of measurements made in 10 cells per muscle.

 
The data shown in Fig. 9 were further analysed by plotting individual profiles for each muscle (not shown) and the area under each curve and above 100% integrated between 50 and 1000 nm. These integrals were then used as summary variables to enable statistical comparison of the diastolic and systolic data (diastolic value=13 205±12 703 units, systolic value=29 468±17 720 units). There was no significant difference between the results at peak systole and at the end of diastole (P0.05, unpaired t-test).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The principal observation of these studies was that there is no detectable transient activation of the Na/K pump current following Na influx via the Na channel in isolated guinea pig myocytes. However, in rabbit myocardium, we present evidence for the existence of a sub-sarcolemmal gradient for Na with Na being highest near the underside of the membrane and falling to levels equal to that in the centre of the cell within approximately 200 nm of the membrane. This sub-sarcolemmal Na gradient was similar whether measured in diastole or systole suggesting, in line with the Na/K pump current measurements, that there is no substantial further elevation of sub-sarcolemmal Na following electrical excitation and the activation of a Na inward current. Thus, while there is clearly a maintained elevation of Na just under the membrane, this sub-sarcolemmal Na does not appear to change between systole and diastole.

In addition to these observations, the measurement and characterisation of the Na pump current in guinea pig myocytes indicates a Km for [K]o activation of 1.6 mM, which is comparable to other recent studies [10,11,29]. Similarly, the Km for [Na]i of 15.5 mM is comparable to those in the literature [10–12] As with other studies, a clear voltage-dependence of Ip was evident, with inward rectification at positive potentials.

Two lines of evidence from this study suggest that, in the unstimulated myocardium, sub-sarcolemmal Na is elevated with respect to bulk Na: (i) in guinea pig myocytes, a significant pump current (Ip) was measured in the absence of pipette Na, and (ii) in rabbit myocardium EPXMA directly indicates a significant elevation of sub-sarcolemmal total Na.

In the stimulated myocardium three lines of evidence suggest that this fuzzy-space Na does not change: (i) there was no detectable transient increase in Na/K pump current immediately after completion of either a single Na current or a train of 20 Na currents, (ii) there was no detectable shift in the reversal potential of INa after 20 consecutive activations when the Na/K pump is functioning normally, and (iii) EPXMA showed no change in the sub-sarcolemmal Na gradient between muscles frozen in systole and those frozen in diastole.

There is evidence that the Na/Ca exchanger and the Na/K pump respond to a common sub-sarcolemmal Na pool [21,30–33]. In addition, previous studies have suggested that the Na/Ca exchanger responds to sub-sarcolemmal Na gradients following Na influx on INa [16,18]. However, since undertaking the present studies, Weber et al. [34] have presented data showing that, in rabbit myocytes, cardiac Na current does not elevate [Na]ss sensed by Na/Ca exchange current. Their data suggest that any influence of INa on [Na]ss, as sensed by the Na/Ca exchanger, is either minimal or is over by the end of the Na inward current (in their case <5 ms). These observations are in agreement with our own in which our temporal resolution is <10 ms after the start of the Na current (i.e. see Fig. 4c) and in which we can detect no transient activation of Na/K pump current immediately after this period. This, of course, does not preclude a more rapid change in [Na]ss during INa which is then rapidly restored to the steady-state condition in less than 10 ms.

The evidence for INa-induced reverse mode Na/Ca exchange is based on the sensitivity of Ca transients to the Na channel inhibitor TTX and the monovalent cation Li during steady-state stimulation. It is therefore possible that these effects relate to the extent of SR Ca load under steady-state conditions and are not due to Ca influx through reverse mode Na/Ca exchange per se (see review by Levi et al. [36]). Subsequent studies in which TTX or Li were applied immediately before stimulation using a rapid solution switcher suggested that Na influx on INa did not induce reverse mode Na/Ca exchange through an elevation of [Na]ss [35,36]. Thus, on this evidence, it is unsurprising that in the present study, INa did not influence Ip.

There are number of possible reasons why, despite a sub-sarcolemmal region of limited diffusion, INa may fail to transiently influence Ip. Firstly, it is possible that the Na influx associated with a single activation of INa may be too small to influence [Na]ss despite this limited sub-sarcolemmal space. Our EPXMA observations, like those of Wendt-Gallitelli et al. [37] would suggest that a diffusional barrier may exist approximately 50 nm below the membrane. Assuming these dimensions, and that our typical cell volume was around 23 pl (estimated from the membrane capacitance measurement of 106 pF according to the method of Satoh et al. for rabbit myocytes [38]) the Na accumulation can be estimated. A single depolarisation would bring in approximately 2x10–16 mol Na ions [39] and, if this were dispersed throughout the entire cell volume, this would increase [Na] by approximately 8 µM. Should all of the Na entering the cell accumulate in a 50 nm wide subsarcolemmal fuzzy-space, then [Na]ss would be elevated by about 0.7 mM. Assuming that bulk cytosolic [Na] is around 8 mM such a rise in sub-sarcolemmal Na can be estimated, from the equation in the legend to Fig. 3, to increase the whole-cell Na/K pump current by about 6 pA—this may not be discernable from the noise in the signal (see Fig. 4). However, 20 consecutive beats should raise [Na]ss by approximately 14 mM. This would increase Na/K pump current by around 82 pA—a level of increase that should easily have been discernable. Since such an increase was not observed then it is likely that most, if not all, of the Na entering the cell during a single stimulus is removed during the diastolic period (even during rapid pacing) and does not progressively accumulate in the cell. Since this diastolic extrusion of Na must occur to maintain ionic equilibrium, there must be a small transient accumulation of Na under the membrane which activates Na extrusion prior to re-establishing [Na]i at its set-point. However, this Na accumulation may be so small as to have no discernable effect on Ip (<5 pA) and no discernable effect on sub-sarcolemmal Na measured 80 ms later using EPXMA.

A second possibility is that there is a transient rise in [Na]ss but, if this is of a magnitude that is sufficient to activate the Na/K pump current, it is complete within the time-course of the Na current itself and a rise in pump current is therefore masked. This possibility, supported by the nearly identical observations of Weber et al. [34] measuring Na/Ca exchange current, allows for the modulation of ion fluxes by the interplay of these transporters without a prolonged change in [Na]ss. While other techniques with more sensitive current measurements or higher precision EPXMA may reveal a transient accumulation of [Na]ss, it is clear from the present study that such an accumulation is likely to be small and/or transient and complete within 10 ms.

If, under these experimental conditions, the majority of Na entering the cell is extruded rapidly during diastole, this contrasts with many studies in multi-cellular preparations that show that rapid pacing induces both a rise in bulk [Na]i and a sustained stimulation of Na/K pump current, that is maintained for the duration of the elevation of [Na]i and, on the termination of stimulation, recovers with a time-course which parallels the recovery of [Na]i [14,40–42]. In the present study, there was neither a transient elevation of Ip within the contractile cycle nor a sustained elevation of basal Ip during rapid pacing. This may be explained by differences between the normothermic, pipette-dialysed, single cell preparation used here and the use of multicellular models often at low temperatures.

One alternative explanation for the failure of INa to transiently stimulate Ip may be related to the spatial geometry of the cardiac myocyte. It is possible that the Na channel, the Na/Ca exchanger and the Na/K pump may not all have equal access to a single homogeneous sub-sarcolemmal compartment. This concept is supported by Wendt-Gallitelli et al. [37] who observed that the sub-sarcolemmal Na gradient was heterogeneous around the sarcolemma and was localised in some regions but not in others. In this regard, it is interesting to note that James et al. [43] have suggested that the {alpha}1 and {alpha}2 isoforms of the Na/K pump may be spatially and functional separate. James et al. [43] claim that their data support the notion that the {alpha}1 isoform (which contributes approximately 80% to the total Na pump current in our cells: unpublished observations), does not influence E–C coupling and Na/Ca exchange. These authors suggest that it is the {alpha}2 isoform (which contributes about 20% to our current measurements) that both sets and responds to [Na]ss in the restricted subsarcolemmal regions associated with the T-tubular system. If such a functional separation is correct, then the failure to see a significant modulation of total pump current in our experiments may be related to the spatial distribution of pump isoforms. However, the validity of the conclusions reached by James et al. [43] have been questioned by Schwartz and Petrashevskaya [44].

Finally, it is important to note, that the EPXMA technique measures total elemental concentration while it is the activity and not the concentration of ions that is the important determinant of the activity of ion transporters. Our interpretation of our EPXMA data assumes that the activity coefficient for Na remains constant from cytosol through the subsarcolemmal space to very close to the membrane. In fact, this seems rather unlikely. Both structured water near the membrane and binding sites on membrane proteins, lipids and sugars may all alter the activity coefficient for Na. If the net effect of this is to decrease the activity coefficient near the membrane then this may offset the rise in total elemental Na. It is also possible that the dynamic buffering imposed by such interactions near the membrane effectively damps subsarcolemmal oscillations in Na ion activity. Such factors, while difficult to quantify, may significantly influence the behaviour of the measured subsarcolemmal gradient and its dynamic contribution to ion fluxes.

In summary, while these experiments support the concept of a sub-sarcolemmal fuzzy-space of limited diffusion of Na, no evidence was found that this sub-membrane Na gradient changes immediately after Na influx via the voltage-gated Na channel or following conditioning trains of stimuli. Any changes in [Na]ss may either be small or transient in nature such that [Na]ss returns to its steady state condition by the termination of INa (i.e. in <10 ms).

Time for primary review: 28 days.


   Acknowledgements
 
This work was supported by grants from The Guy's and St Thomas’ Charitable Foundation (MJS and BdZS) and The Wellcome Trust (MJS and AW). The help and advice of Jules Hancox is also gratefully acknowledged.


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

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