Cardiovascular Research

Cardiovascular Research 2003 57(4):986-995; doi:10.1016/S0008-6363(02)00848-9
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

[Na⁺]_i and the driving force of the Na⁺/Ca²⁺-exchanger in heart failure

A Baartscheer^*, C.A Schumacher, C.N.W Belterman, R Coronel and J.W.T Fiolet

Experimental and Molecular Cardiology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

a.baartscheer{at}amc.uva.nl

^* Corresponding author. Laboratory of Experimental Cardiology, Room M-0-052, Academic Medical Center, University of Amsterdam, P.O. Box 22700, Meibergdreef 9, 1100 DE Amsterdam, The Netherlands. Tel.: +31-20-566-3265; fax: +31-20-697-5458.

^* For this manuscript Professor A. Fabiato acted as guest editor.

Received 5 June 2002; accepted 6 December 2002

	Abstract

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
Objective: Diastolic calcium is increased in myocytes from failing hearts despite up-regulation of the principal calcium extruding mechanism the Na⁺/Ca²⁺-exchanger (NCX). We hypothesize that increased diastolic calcium ([Ca²⁺]_i) is secondary to increased cytosolic sodium ([Na⁺]_i) and decreased driving force of NCX (G_exch). Methods: The stimulation rate dependence of simultaneously measured cytosolic sodium ([Na⁺]_i), calcium transients ([Ca²⁺]_i) and action potentials were determined with SBFI, indo-1 and the perforated patch technique in midmural left ventricular myocytes isolated from rabbits with pressure and volume overload induced heart failure (HF) and in age matched controls. Dynamic changes of G_exch were calculated. Results: With increasing stimulation frequency, 0.2–3 Hz (all data HF versus control): [Na⁺]_i increased (6.4 to 10.8 versus 3.8 to 6.4 mmol/l), diastolic [Ca²⁺]_i increased (142 to 219 versus 47 to 98 nmol/l), calcium transient amplitude decreased in HF (300 to 250 nmol/l) but increased in control (201 to 479 nmol/l), action potential duration (APD₉₀) decreased (380 to 260 versus 325 to 205 ms) and time averaged G_exch decreased (6.8 to 2.8 versus 8.7 to 6.4 kJ/mol. With increasing stimulation rate the forward mode time integral of G_exch decreased in HF by about 30%, the reversed mode time integral increased about ninefold and the duration of reversed mode operation more than sixfold relative to control. Conclusions: [Na⁺]_i is increased in HF and the driving force of NCX is decreased. NCX exerts thermodynamic control over diastolic calcium. Disturbed diastolic calcium handling in HF is due to decreased forward mode G_exch secondary to increased [Na⁺]_i and prolongation of the action potential. Enhanced reversed mode G_exch may account for increased contribution of NCX to e–c coupling in HF.

KEYWORDS Calcium (cellular); Heart failure; Myocytes; Na/Ca-exchanger

	1. Introduction

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
Disturbed calcium handling underlies cardiac contractile failure and arrhythmogenesis in the hypertrophic and failing heart. The electrogenic Na⁺/Ca²⁺-exchanger (NCX) is the major sarcolemmal calcium transport system regulating cytosolic calcium. Due to the electrogenic nature of NCX, carrying either inward current in forward mode (V_m<E_rev-NCX) or outward current in reversed mode (V_mE_rev-NCX), it modulates action potential configuration and duration and plays a role in the genesis of delayed after depolarizations (DADs). Consequently, altered activity could contribute to contractile dysfunction and arrhythmogenesis.

During the cardiac cycle NCX most of the time operates in forward mode (calcium extrusion). Up-regulation of NCX on the mRNA and protein level would be expected to promote net loss of Ca²⁺ from the cell leading to decreased diastolic calcium [Ca²⁺]_i, because fluxes are basically proportional to protein expression. Indeed, acute adenovirus induced over-expression of NCX in the rat and the rabbit causes reduction of diastolic [Ca²⁺]_i [1] and SR calcium content [2]. In human end-stage heart failure and in animal models of heart failure increased expression and/or activity of NCX have been reported (reviewed in Ref. [3]). Nevertheless, increased expression of NCX in heart failure was not associated with decreased diastolic calcium, but rather with either unchanged [4] or increased diastolic calcium [5,6].

The activity of NCX depends not only on the kinetic properties or expression level, but also on the magnitude and dynamic change of the driving force (G_exch) during the cardiac cycle. G_exch is a function of [Na⁺]_i, [Ca²⁺]_i, and trans-membrane potential (V_m); relative to [Ca²⁺]_i, [Na⁺]_i is quantitatively of major importance, because it contributes to G_exch with the third power [7]. Increased [Na⁺]_i has been reported in hypertrophied myocytes [8]. So far data on [Na⁺]_i in heart failure are scarce [9,10]. A more detailed very recent study demonstrates increased [Na⁺]_i in HF [11], which was mainly attributed to a TTX sensitive mechanism rather than to up-regulated or altered sarcolemmal Na⁺ transport mechanisms such as increased Na⁺/H⁺-exchange (NHE) activity [12] or decreased Na⁺/K⁺-ATPase activity [13]. Regardless of the responsible mechanism, increased [Na⁺]_i would cause a shift of G_exch to lower values in forward and higher values in reversed mode. In addition, prolonged action potential duration [14] and reduced calcium transient amplitude [15] in HF would contribute to a change of the G_exch profile during the cardiac cycle.

This study aims to establish (1) how altered [Na⁺]_i, [Ca²⁺]_i and action potential configuration in HF modify G_exch and (2) how this could explain disturbed calcium handling.

	2. Methods

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
2.1 The rabbit model of pressure and volume overload induced heart failure and isolation of left midmural ventricular myocytes
Animal care and handling conformed to Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996) and the study was approved by the local ethical committee.

HF was induced in eight rabbits (New Zealand White, SPF, 3–3.5 kg) by combined volume and pressure overload as described previously [14,16]. Volume overload was produced by aortic valve rupture increasing pulse pressure by 100% and pressure overload was produced 3 weeks later by a suprarenal abdominal aortic banding to approximately 50%. After 12 weeks LVEDP was measured, animals were sacrificed, hearts were isolated. Body weight, heart and lung weight were measured and presence of ascites was documented as described previously [14,17]. Age matched untreated animals (n=6) served as controls. Midmural left ventricular myocytes were isolated as described previously [18] and stored until use at room temperature in separate vials, each containing about 10⁵ myocytes in 5 ml solution containing (mmol/l): [Na⁺] 156, [K⁺] 4.7, Ca²⁺ 2.6, [Mg²⁺] 2.0, [Cl^–] 150.6, [HCO₃^–] 4.3, [HPO₄^2–] 1.4, [Hepes] 17, [glucose] 11 supplied with 1% fatty acid free albumin (pH 7.3). Cell dimensions of 100 randomly chosen rod shaped myocytes were determined.

2.2 Measurement of free cytosolic [Ca²⁺]_i, [Na⁺]_i and trans-membrane potentials
Before each individual experiment, cells were loaded during 30 min with 5 µmol/l indo-1/AM or 120 min with 10 µmol/l SBFI and 0.01% pluronic, washed twice with fresh Hepes solution (without albumin), and kept for another 15 min to ensure complete de-esterification. [Ca²⁺]_i and action potentials were measured simultaneously. Hardware for data recording consisted of a patch clamp amplifier (Axopatch 200B), two laboratory-made differential amplifiers for photomultiplier signals and a combined A/D and D/A board (DAS1802AO, Keithley Metrabyte) controlled by custom made software (Test-point).

Myocytes were placed in a thermally controlled (37 °C) cell chamber on the stage of an inverted fluorescence microscope (Nikon Diaphot) and superfused with the same Hepes buffered solution as above (without albumin) at a rate of 1–2 ml/min. A quiescent rod-shaped myocyte was selected, stimulation was started and a rectangular diaphragm was used to restrict the fluorescence measuring area to the myocyte surface. Dual wavelength emission of Indo-1 was recorded (410/516 nm, excitation at 340 nm) and cellular free [Ca²⁺]_i was calculated and calibrated as described previously [19]. Fig. 1 shows representative examples of raw data and processing of signals. Signals were corrected for background recorded from indo-1 free myocytes (about 10% of raw signals). A second correction (allowing for mitochondrial compartmentalization of indo-1 (37%, see Ref. [19]) and stimulation rate dependence of mitochondrial calcium [20] was applied, in order to obtain cytosolic free [Ca²⁺]_i as described previously [19]. In parallel experiments dual wavelength emission of SBFI was recorded (516/590 nm, excitation at 340 nm), signals were corrected for background fluorescence recorded from SBFI free myocytes and [Na⁺]_i was calculated and calibrated as described previously [21]. Action potentials were recorded using the perforated patch-clamp technique with a pipette solution containing (mmol/l): [Na⁺] 6 or 10 (in control or HF myocytes, respectively), [K⁺] 140, [Mg²⁺] 1.0, [Cl^–] 153.6, [HPO₄^2–] 1.4, [Hepes] 17, [glucose] 11 [Ca²⁺] 2.6 and 0.2 mg/ml amphotericin B (pH 7.1). Pipette resistance was 3–5 M. The potential to bath solution was adjusted to zero. Capacitance and the pipette series resistance were compensated to about 80%. Access resistance to the cell decreased within 10 min after seal formation. Fluorescence and action potential signals were digitized at 1 kHz.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Representative examples in a HF myocyte stimulated at 2 Hz of measured raw fluorescence signals, ratio data, cellular [Ca2+]i and calculated ‘true’ cytoplasmic [Ca2+]i. Respective background fluoresce signals were 0.15 at 410 nm and 0.10 at 516 nm. Rmax, Rmin, β and kd were 4.5, 0.55, 2.1 and 250 nM, respectively. Mitochondrial compartmentation 37% (see Ref. [19]).

 
Stimulation rate dependence (0 to 3 Hz) of [Na⁺]_i, [Ca²⁺]_i and action potentials was studied in each individual myocyte after 2 min of conditioning at 2 Hz. To obtain steady state conditions also a 2 min period of stimulation preceded measurements at any frequency.

2.3 Formulation of G_exch

in which V_m is membrane potential, F the Faraday constant and R and T have their usual meaning. In this study positive values of G_exch correspond to driving force in forward mode (inward sodium transport) and negative values to reversed mode (outward sodium transport), respectively.

2.4 Statistics
Data are expressed as mean±S.E.M. for six control rabbits (18 myocytes) and eight HF rabbits (24 myocytes), three myocytes per heart. Values for myocytes of individual hearts were averaged. Two-way analysis of variance (ANOVA; with a post hoc test according to Student–Newman–Keuls) or Student's t-test was used to test for statistical significance where appropriate at a level of significance of P<0.05.

	3. Results

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
3.1 Animal characteristics
Table 1 summarizes the parameters relevant to HF rabbits and age matched controls. Bodyweight was not different in HF and control. In HF, heart and lung weights relative to bodyweight and LVEDP were significantly increased. Ventricles were hypertrophied and dilated. Ascites was found in three out of eight HF rabbits. The average maximal myocyte length and width (largest cross sectional diameter) were significantly increased in HF myocytes (t-test).

View this table: [in this window] [in a new window]   Table 1 Animal characteristics

 
3.2 [Na⁺]_i, [Ca²⁺]_i and trans-membrane potentials
[Na⁺]_i did not show cycle related fluctuations in either group of animals. Fig. 2 summarizes the average [Na⁺]_i as a function of stimulation frequency (0 to 3 Hz). At all stimulation rates [Na⁺]_i was significantly higher in HF than in control (ANOVA). [Na⁺]_i increased with stimulation rate from 6.4 to 10.8 mmol/l in HF and from 3.8 to 6.1 mmol/l in control. The difference between [Na⁺]_i at 0 Hz and 3 Hz was significantly larger in HF than in control (t-test P<0.0001).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Stimulation rate dependence of cytosolic [Na+]i. Control (open circles) and HF (closed circles).

 
Fig. 3 shows the stimulation rate dependence of diastolic [Ca²⁺]_i (left panel) and calcium transient amplitude (right panel). At all stimulation rates diastolic [Ca²⁺]_i was significantly higher in HF than in control (ANOVA). With increasing stimulation rate diastolic [Ca²⁺]_i increased significantly more in HF than in control (t-test, P<0.0004), the calcium transient amplitude remained constant in HF with a tendency to decrease, but increased with stimulation rate in control. Kinetic parameters of calcium transients are summarized in Table 2. With increasing frequency time to peak and calcium transient duration decreased. Time to peak was not different in HF and control, but calcium transient duration was significantly prolonged at all frequencies (ANOVA).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Stimulation rate dependence of diastolic [Ca2+]i (left panel) and calcium transient amplitude (right panel). Control (open circles) and HF (closed circles).

 

View this table: [in this window] [in a new window]   Table 2 Calcium transient kinetic characteristics

 
Fig. 4 shows representative examples of simultaneously measured action potentials and calcium transients. In HF action potential duration was prolonged, diastolic [Ca²⁺]_i was higher, calcium transient amplitude was smaller and relaxation rate was slowed compared to control.

View larger version (13K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Representative examples of action potentials (left panel) and calcium transients (right panel) measured simultaneously in a control (solid lines) and a HF (dotted lines) myocyte stimulated at 2 Hz.

 
Fig. 5 shows that action potential duration at 90% repolarization (APD₉₀) decreased with increasing stimulation frequency in HF and control. APD₉₀ was significantly prolonged at any frequency (ANOVA) in HF relative to control by about 50 ms.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Stimulation rate dependence of action potential duration measured at 90% repolarization (APD90). At any frequency APD90 was significantly larger in HF than in control (P<0.03).

 
3.3 The driving force of the Na⁺/Ca²⁺-exchanger (G_exch)
The dynamic change of G_exch during the cardiac cycle was calculated from [Na⁺]_i, [Ca²⁺]_i and the trans-membrane potential in each individual myocyte. Fig. 6 shows representative examples of G_exch profiles in a steady state 2 Hz stimulated control (left panel) and HF (middle panel) myocyte. In HF forward mode G_exch was shifted to less positive values and duration was reduced, reversed mode G_exch was shifted to more negative values (dotted area) and duration was substantially prolonged relative to control. In control myocytes an overshoot was observed in forward mode G_exch upon repolarisation, due to the difference between action potential and calcium transient duration. In HF, where action potential and calcium transient duration approached each other, the overshoot was greatly reduced. The right panel summarizes the stimulation rate dependence of time averaged G_exch (averaged over the entire cardiac cycle; solid lines in the two left panels). Time averaged G_exch was significantly smaller in HF than in control at all frequencies (ANOVA) and decreased more (t-test, P<0.0001). Forward and reversed mode both contribute to time averaged G_exch. The respective surface areas are representative measures of these contributions. Fig. 7 shows the stimulation rate dependence of the forward mode (top left) and reversed mode (top right) surface areas. At all frequencies the forward mode surface area in HF was less and the reversed mode surface area was larger than in control. The bottom panels show the change of the respective surface areas in HF relative to control; forward mode was reduced frequency dependently from 0.85 to 0.45, and reversed mode was enhanced about nine fold over the entire range of frequencies. Consequently, calcium efflux becomes reduced, and calcium influx during the action potential becomes increased in HF relative to control.

View larger version (29K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 The dynamic change of Gexch and the time averaged Gexch during one cardiac cycle. Dynamic change of Gexch in a 2 Hz stimulated control (left panel) and HF (middle panel) myocyte. The dotted area corresponds to reversed mode operation of NCX. The solid lines represent Gexch time averaged over one cardiac cycle. The stimulation rate dependence of the time averaged Gexch (right panel). Control (open circles) and HF (closed circles).

 

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 Stimulation rate dependence of forward and reversed mode contributions to time averaged Gexch. Forward mode surface area during the cardiac cycle (upper left). Reversed mode surface area during the cardiac cycle (upper right). Control (open circles) and HF (closed circles). The respective surface areas in HF relative to control for forward mode (bottom left) for reversed mode (bottom right). Note the different y-axis scaling for control and HF in the upper right panel.

 
[Na⁺]_i, [Ca²⁺]_i and action potential configuration all contribute to the difference in time averaged G_exch between HF and control (G_exch-HF–G_exch-ctrl). Fig. 8 shows the frequency dependence of the individual contributions of [Na⁺]_i, [Ca²⁺]_i and V_m. Increased of [Na⁺]_i contributes most, but is hardly stimulation rate dependent. Altered action potential contributes particularly at the higher stimulation rates, but much less than [Na⁺]_i. Altered diastolic [Ca²⁺]_i and calcium transient configuration partly compensate for the effects of increased [Na⁺]_i and action potential, but more at the lower stimulation frequencies. For clarity the open squares show the combined effects [Na⁺]_i, [Ca²⁺]_i and V_m.

View larger version (22K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 8 Stimulation rate dependence of individual parameters contributing to the difference between Gexch in HF and control (open squares). Contribution of [Na+]i (closed triangles), contribution of diastolic [Ca2+]i and calcium transient (closed squares), contribution of trans-membrane potential (closed circles).

 

	4. Discussion

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
This study quantifies the impact of altered sodium homeostasis in left ventricular myocytes from hearts of rabbits with pressure and volume overload induced HF on dynamic changes of the driving force of NCX (G_exch) and calcium handling. We demonstrate that in HF frequency dependently [Na⁺]_i and diastolic [Ca²⁺]_i are increased, action potential is prolonged and that G_exch is shifted to less positive values during diastole and to more negative values during systole. These changes are stimulation rate dependent. Besides prolongation of the action potential, increased [Na⁺]_i contributes most to this shift, while increased diastolic [Ca²⁺]_i and altered calcium transient configuration have a compensatory effect. We conclude from the data presented that, despite up-regulation of NCX in heart failure (for a review, see Ref. [3]), the decreased driving force, secondary to increased [Na⁺]_i is causal to increased diastolic [Ca²⁺]_i and associated disturbed diastolic function.

We used a well established model of combined volume and pressure overload induced heart failure in rabbits as described previously [14,16]. This model of heart failure has been widely and successfully used over the last decade. Advantages of the model are (1) the reproducible development of failure within 2 to 3 months with all features also clinically observed in humans (hypertrophy, dilatation, dispnoe, decreased diastolic function and arrhythmogenesis) [17,22] and (2) the greater similarity in action potential characteristics and excitation–contraction coupling between human and rabbit than other models of heart failure such as, e.g., a rat model.

4.1 [Na⁺]_i,diastolic [Ca²⁺]_iand action potential prolongation in HF
Increased [Na⁺]_i in hypertrophy has been reported previously [8], but so far data on [Na⁺]_i in heart failure are scarce [9,10]. Very recently, a more detailed study in a similar model of HF reported increased [Na⁺]_i, which was attributed mainly to a TTX sensitive mechanism, presumably Na-channel activity, but little contribution of Na⁺/H⁺-exchange (NHE) and no involvement of Na⁺/K⁺-ATPase [11]. Quite intriguingly, these data were obtained in quiescent myocytes where both Na-channel and NHE activity are supposed to be minimal. Our [Na⁺]_i data at rest are about 50% less than those of Despa et al. [11] both in control and HF. Our previous data [9,10] and unpublished observations (submitted) rather suggest a key role for NHE and no involvement of altered Na-channel dependent sodium influx. This would agree with data on up-regulated activity but unchanged expression of NHE [12,23] and on unchanged sodium channel kinetics in HF [24], although calcium overload induced calmodulin dependent modulation of slow inactivation of the Na-channel in HF can not be excluded [25]. Also reduced sodium efflux secondary to depressed Na⁺/K⁺-pump activity should be considered [13].

Prolongation of the action potential is commonly found in heart failure (for a review, see Ref. [26]). We measured prolongation of action potential duration, which was rather independent of stimulation rate (Fig. 4), which contrasts with other studies reporting convergence with increasing stimulation rate [14,26,27]. It may be speculated that methodological differences underlie the discrepant results. We used pipette sodium concentrations corresponding to those found in HF and control in the perforated patch technique, which causes minimal disturbance of the intracellular milieu, in contrast with conventional micro-electrodes [14] or the whole cell patch clamp technique [27] in which such disturbance can not be excluded.

The decreased electrochemical potential of Na⁺ across the sarcolemma caused by the increased [Na⁺]_i, reduces the forward driving force of NCX, and causes increased steady state diastolic [Ca²⁺]_Ii, which is confirmed by the inter-dependence of [Na⁺]_i and diastolic [Ca²⁺]_i shown in Fig. 3. Increased diastolic [Ca²⁺]_i in HF in combination with down regulation of the SR Ca²⁺-ATPase (SERCA) [28] interferes with SR calcium handling. On the one hand, down regulation of SERCA may underlie decreased SR calcium loading in particular at the higher stimulation rates explaining at least in part the negative (or flat) relationship between the calcium transient amplitude and stimulation rate (Fig. 3) [29]. On the other hand, increased diastolic [Ca²⁺]_i contributes to this by affecting open probability of SR calcium release channels (RyR). This has been experimentally demonstrated [30] and is consistent with the observation that spontaneous calcium spark incidence increased with elevation of [Ca²⁺]_i from 100 to 150 nmol/l [31]. Abnormal SR calcium handling and RyR conductance in HF is also implicated in DAD related arrhythmogenesis in HF [32,33]. Spontaneous SR calcium release may generate DADs by inward current carried by up-regulated NCX [22].

4.2 G_exch and activity of NCX in heart failure
Up-regulation and/or increased activity of NCX are commonly found in both human end-stage heart failure and in animal models [3]. For the animal model used in this study NCX was up-regulated by about 93% [4] without an effect on diastolic calcium. Up-regulation of NCX by itself would be expected to decrease diastolic calcium, which indeed has been demonstrated in adenovirus induced acute over-expression of NCX [1]. On the other hand transgenic mice over-expressing NCX [34] no change of diastolic calcium was observed. Despite up-regulation of NCX, the present results as well as other studies [5,6] report increased diastolic [Ca²⁺]_i in HF. A reduced forward driving force of NCX might explain this seemingly contradictory result.

Therefore, the shift of G_exch to less positive values in HF, its dependence on stimulation rate (Fig. 6) and the individual contributions of [Na⁺]_i. [Ca²⁺]_i and action potential to this shift (Fig. 8) are central to this study. The physiological implications of the downward shift of G_exch are: (1) increased and prolonged reversed mode activity during the action potential and enhanced calcium influx during the cardiac cycle in addition to I_CaL. This might enhance e–c coupling to compensate for loss of contractility associated with down regulation of SERCA. With little reversed mode, such as in control myocytes, NCX hardly contributes to e–c coupling [35,36]. However, enhanced reversed mode NCX can trigger calcium release from SR [1,19], which is in agreement with a recent model study that indicated a steep rise in SR triggering efficiency to about 25% by NCX when [Na⁺]_i was increased to 10 mmol/l [37]. Recent reports on increased relative SR depletion during systole in HF are consistent with this model study [29,37]. (2) A stimulation rate dependent progressive increase of diastolic calcium in HF (Figs. 3 and 5). (3) In order to maintain calcium homeostasis in steady state, not only calcium channel related but also the additional NCX reversed mode related calcium influx should be compensated for at a lower forward mode time averaged G_exch. This might explain the necessity for molecular up-regulation of NCX in HF.

4.3 Limitations of the study
We took maximal cell length and width as a measure of the degree of cellular hypertrophy. Obviously such an approach precludes estimation of changes of cell volume. In addition we can not entirely exclude that a small fraction of the myocyte population consisted of still side to side coupled cell pairs. However, it seems reasonable to assume that there would be no difference between HF and control in this respect.

In vivo resting heart rate of rabbits is about 2.5–3 Hz, which may increase to well above 4 Hz with exercise and/or stress. It is common experience that in isolated control rabbit myocytes the maximal stimulation rate is around 4 Hz. In HF myocytes, with prolonged action potential and refractory period, maximal stimulation rate is even less. Absence of adrenergic regulation in isolated myocytes might explain the difference with the in vivo situation. Indeed, application of noradrenalin to myocytes enhances maximal stimulation rates. Although some HF myocytes could be stimulated up to 3.5 Hz, the majority of HF cells failed to respond to such a rate. Therefore, this study can not provide information on sodium and calcium handling at higher, more physiological stimulation rates.

We used bulk cytosolic [Na⁺]_i and [Ca²⁺]_i data to calculate G_exch. It may be argued that the actual driving force of NCX could deviate from calculated G_exch due to the existence of sub-sarcolemmal gradients of these ions. So far, there is few data on the actual magnitude of sub-sarcolemmal sodium and the quantitative importance is disputed. The existence of sub-sarcolemmal calcium gradients has been demonstrated using optical [38] and electrophysiological [39] techniques; estimates for maximal systolic sub-sarcolemmal [Ca²⁺]_i were up to four-times bulk values [39]. Although ‘fuzzy’ space effects would thus affect our calculations quantitatively, this is not necessarily so qualitatively.

Some recent studies suggest a that the Na:Ca stochiometry for NCX might be higher than 3:1 used in our calculations of G_exch [40,41]. This would not only implicate that all our G_exch data should be scaled up by at least 6 kJ/mol, but also that NCX would never operate in reversed mode in physiological conditions and that the diastolic reversal potential would much more positive than usually measured values around –20 mV.

Very recently, a study was published by Pieske et al., which corroborates the findings in this study regarding frequency dependence and elevation of intracellular sodium in human myocardium of patients with heart failure [42].

	5. Conclusions

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 
Increased [Na⁺]_i and prolonged duration of action potential cause a decrease of G_exch in HF and increased duration the Na⁺/Ca²⁺-exchanger is in reversed mode. This results in an increase of diastolic calcium despite an up-regulated Na⁺/Ca²⁺-exchanger and might be a prerequisite for arrhythmogenesis in HF.

Time for primary review 36 days.

	Acknowledgements

 
This work was supported by The Netherland Heart Association (96039).

	References

Top Abstract 1. Introduction 2. Methods 3. Results 4. Discussion 5. Conclusions References

 

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