Cardiovascular Research

Cardiovascular Research 2003 57(4):996-1003; doi:10.1016/S0008-6363(02)00829-5
© 2003 by European Society of Cardiology

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Copyright © 2003, European Society of Cardiology

The functional effect of adenoviral Na⁺/Ca²⁺ exchanger overexpression in rabbit myocytes depends on the activity of the Na⁺/K⁺-ATPase

W Schillinger^*, A Ohler, S Emami, F Müller, C Christians, P.M.L Janssen, H Kögler, N Teucher, B Pieske, T Seidler and G Hasenfuss

Herzzentrum Göttingen, Kardiologie und Pneumologie, Georg-August-Universität Göttingen, Robert-Koch-Strasse 40, 37075 Göttingen, Germany

^* Corresponding author. Tel.: +49-551-39-6349; fax: +49-551-39-9804. schiwolf{at}med.uni-goettingen.de

Received 31 May 2002; accepted 26 November 2002

	Abstract

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

 
Objectives: The functional consequences of Na⁺/Ca²⁺ exchanger (NCX) overexpression in heart failure have been controversially discussed. NCX function strongly depends on intracellular sodium which has been shown to be increased in heart failure. Methods and results: We investigated the Na⁺/K⁺-ATPase (NKA) inhibitor ouabain (0.5–16 µmol/l) in electrically stimulated, isotonically contracting adult rabbit cardiocytes overexpressing NCX after adenoviral gene transfer (Ad-NCX-GFP, 48 h culture time). Myocytes transfected with adenovirus encoding for green fluorescent protein (Ad-GFP) served as a control. Contractions were analyzed by video-edge detection. In the Ad-NCX-GFP group, the maximum inotropic response was significantly reduced by 50.7% (P<0.05). This was a result of an enhanced susceptibility to contracture after exposure to the drug (median concentration (25–75%): 4 (4–8) vs. 8 (6–16) µmol/l, P<0.05). When analyzing relaxation before contracture, the maximum relaxation velocity was reduced (0.15±0.04 vs. 0.27±0.04 µm/s, P<0.05) and the time from peak shortening to 90% of relaxation was increased (298±39 vs. 185±15 ms, P<0.05). No differences in systolic and diastolic parameters were observed with the Na⁺ channel modulator BDF9198 (1 µmol/l). Conclusions: Inhibition of NKA by ouabain induces a combined diastolic and systolic dysfunction in NCX overexpressing rabbit myocytes. This may be the consequence of cytoplasmic Ca²⁺ overload due to inhibition of forward mode or induction of reverse mode Na⁺/Ca²⁺ exchange. In end-stage failing human myocardium and during digitalis treatment this mechanism may be of major importance.

KEYWORDS Calcium (cellular); Contractile function; Heart failure; Na/Ca-exchanger; Na/K-pump

	1. Introduction

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

 
Recently, increased expression and function of the Na⁺/Ca²⁺ exchanger has been blamed for playing an important role in the mechanisms underlying the disturbed myocardial function of the end-stage failing heart in humans [1–5] as well as in animal models of heart failure [5–7]. In many species including humans, Na⁺/Ca²⁺ exchange in its forward mode is believed to consist the main route for Ca²⁺ extrusion across the sarcolemma under physiological conditions in a stoichiometry of three Na⁺ ions that enter the cell for one Ca²⁺ ion eliminated from the cytoplasm [8–10], although recently other authors suggested a ratio of 4:1 [11]. During steady-state contractions, Ca²⁺ entry by sarcolemmal Ca²⁺ channels usually compensates for Ca²⁺ elimination by Na⁺/Ca²⁺ exchange keeping intracellular Ca²⁺ concentration as well as sarcoplasmic reticulum Ca²⁺ load constant [12,13]. During the early phase of the action potential, when the membrane is depolarized to values positive to the reversal potential the Na⁺/Ca²⁺ exchanger may however also work in a reverse mode promoting Ca²⁺ influx and Na⁺ extrusion [5,8–10].

In a recent study we were able to demonstrate that overexpression of Na⁺/Ca²⁺ exchanger in healthy adult rabbit myocytes following adenoviral gene transfer diminished SR Ca²⁺ load and systolic myocardial performance [14]. We found that electrically stimulated, isotonically contracting myocytes exhibited a reduction in basal fractional shortening and a blunted frequency-dependent potentiation of shortening. Furthermore, reduced shortening was associated with reduced caffeine-induced contractures, indicating reduced SR Ca²⁺ load. It has been speculated that more exchanger molecules in the NCX-transfected cells temporarily cause trans-sarcolemmal Ca²⁺ efflux to exceed Ca²⁺ influx until a new steady state is reached at a lower SR Ca²⁺ load. Consequently, SR Ca²⁺ release and reuptake are lower in NCX-transfected myocytes compared to control cells resulting in reduced activation of contractile proteins and reduced fractional shortening. In accordance, a previous study in cardiac trabeculae from end-stage failing human hearts had demonstrated systolic contractile dysfunction in hearts with increased Na⁺/Ca²⁺-exchanger expression but unchanged SR-Ca²⁺-ATPase [15]. Thus, an increase in the expression of the Na⁺/Ca²⁺ exchange protein may result in an increased proportion of molecules functioning in the forward mode which then results in cellular Ca²⁺ loss.

However, the function of the Na⁺/Ca²⁺ exchanger is not only determined by protein levels, but is strongly dependent on the transmembrane gradients for Na⁺ and Ca²⁺. A rise in [Na⁺]_i is believed to result in inhibition of the forward mode or induction of the reverse mode [5,8–10]. In human heart failure, activity and expression of the Na⁺/K⁺-ATPase is reduced which predicts an increase in [Na⁺]_i [16–18]. Other sarcolemmal Na⁺-transporting systems like the Na⁺-channel or the Na⁺/H⁺ exchanger may as well influence [Na⁺]_i. Accordingly, increased [Na⁺]_i has been measured in multicellular muscle strip preparations and isolated myocytes from end-stage failing human myocardium and animal models of heart failure [19–21]. We therefore sought to investigate the effect of Na⁺/K⁺-ATPase inhibition by ouabain on contractile function in cultured rabbit myocytes overexpressing the Na⁺/Ca²⁺ exchanger following adenoviral gene transfer. In addition, the functional consequences of the Na⁺-channel modulator BDF9198 were analyzed in both cell types.

	2. Methods

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

 
2.1 Material
The Na⁺-channel modulator BDF9198 was a generous gift from Professor Mest (Lilly, Hamburg). All other chemicals indicated were purchased from commercial suppliers with highest purity grade available.

2.2 Recombinant adenoviruses
A recombinant replication deficient type 5 adenovirus carrying the canine full-length Na⁺/Ca²⁺ exchanger gene (NCX) was kindly supplied by Ken Philipson and upscaling was conducted as described [14,22]. Expression of Na⁺/Ca²⁺ exchanger is driven by the constitutive active cytomegalovirus promoter (CMV). The virus also encodes for the wild type green fluorescent protein (GFP) as a reporter gene expressed separately under the control of a separate CMV promoter (Ad-NCX-GFP). Recombinant adenovirus carrying the CMV-driven GFP (pQBI-Ad-GFP) was used as a control. Plasmid vectors were purchased from Q-biogene (Illkirch, France).

2.3 Primary culture of rabbit ventricular myocytes and adenovirus infection
Chinchilla bastard rabbits (2.0–2.5 kg) were used for myocyte experiments. All procedures conformed with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). The animals were heparinized and anesthetized with sodium thiopental (50 mg/kg i.v.). The heart was rapidly removed and mounted to a modified Langendorff perfusion setup. The digestion was performed with Tyrode solution (25 µmol/l Ca²⁺) containing collagenase type II (250 U/ml) and protease type XIV (0.04 mg/ml) as recently described [14]. Atria were cut off and the ventricles were immersed in Tyrode solution containing 20 mmol/l 2,3-butanedione monoxime, and 50 µmol/l Ca²⁺. The heart was cut into chunks and myocytes were freed by four rounds of mincing and gentle manual agitation. The myocytes were filtered through sterile nylon gauze (200 µm mesh) and progressively exposed to increasing Ca²⁺ concentrations in Tyrode solution. Myocytes were counted and adenoviral infection was performed with indicated multiplicity of infection (MOI). Myocytes were plated at a density of 5x10⁵ rod-shaped cells/ml onto laminin (10 µg/ml)-coated tissue culture dishes. After 2 h, unattached cells were removed by three wash steps and myocytes were cultured for 48 h prior to analysis in supplemented M199 medium. The verification of the transgene expression and virus transfection efficiency was performed by Western immunoblots and reverse transcriptase polymerase chain reaction with different MOIs as described [14]. In addition, infection efficiency with Ad-NCX-GFP at different MOI was monitored by fluorescence microscopy at an excitation wave length of 480 nm. A total of 500 cells were counted in each culture dish to allow for quantification of fluorescent cells.

2.4 Myocyte shortening measurements
Cardiomyocytes plated on laminin-coated 35 mm Petri dishes were placed in a custom-made, heat-thermostated chamber on a Nikon microscope stage. Myocytes from eight different animals transfected with adenoviruses encoding for NCX and GFP were used for shortening measurements. Only one myocyte was randomly selected in each culture dish. Myocytes were superfused at a flow rate of 2.5–3 ml/min with Krebs–Henseleit solution (KHS, 37 °C, pH 7.4, bicarbonate buffered, containing 1.75 mmol/l Ca²⁺) in equilibrium with 95% O₂/5% CO₂. Cells were electrically stimulated by field stimulation at a frequency of 1 Hz and myocyte shortening was measured by a video edge-detection system (Crescent Electronics) at a sampling rate of 240 Hz. On- and off-line analysis was performed with custom-designed Labview software (National Instruments). Cells selected for data analysis had clear striation, rod-shaped form and a 1:1 pacing capture. Only myocytes that showed stable resting cell length during basal steady-state shortening were included into further analysis. Steady state fractional shortening was defined as no additional change in fractional shortening of more than 5% in 30 s. Then, dose-dependence of mechanical parameters from ouabain (0.5, 1, 2, 4, 8, 16 µmol/l) was analyzed at steady-state shortening for each concentration. Fractional shortening at increasing ouabain concentrations was defined relative to the steady state resting cell length at each ouabain concentration. Usually steady state fractional shortening at increasing ouabain concentrations was reached within 6–10 min. For a given concentration, myocytes lost their rod-shaped form and went into contracture. Some myocytes exhibited steady state conditions before the contracture occurred, thus allowing recording of contractile parameters at the very same ouabain concentration that was noted as contracture concentration. Sometimes, resting cell length and fractional shortening continuously decreased and the contracture developed gradually following an increase of ouabain. In this case, no steady state conditions were reached at that given concentration. Furthermore, a series of experiments was performed in the presence of the Na⁺-channel modulator BDF9198 at a concentration of 1 µmol/l and otherwise identical conditions.

2.5 Statistical analysis
Data are presented as mean±S.E.M. Dose–response measurements were analyzed by two-way repeated measures ANOVA followed by Student–Newman–Keul's test where appropriate. Differences in medians (Fig. 4) were analyzed by rank-sum test. The effect of BDF9198 was analyzed by unpaired t-test. A P<0.05 was accepted as statistically significant.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 Box plot demonstrating the concentration at which the cells developed a contracture and got round-shaped. Medians are indicated with crosses. The median of the GFP-group was significantly different from the median of the NCX group (P<0.05). Boxes represent 25–75% confidence intervals while error bars indicate 10–90% confidence intervals.

 

	3. Results

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

 
3.1 Transfection efficiency
All adenovirus preparations (Ad-NCX-GFP and Ad-GFP) were tested for transfection efficiency at different MOIs before use. In order to verify the efficiency, we counted the percentage of cultured myocytes that exhibited green fluorescence at 48 h after viral infection with a MOI of 10 plaque forming units (pfu)/cell. Only virus that yielded an efficiency of <90% at a MOI of 10 was used for further experiments. Thus, there was only small variability with respect to different virus preparations and different culture dishes, respectively. This was similar to the findings in a previous work from our group [14]. The Ad-GFP virus was further used as a negative control to exclude that functional effects may result from the virus vector or the expression of the green fluorescent protein itself. We found a virus dose dependent increase in the expression of the Na⁺/Ca²⁺ exchanger as verified by RT-PCR and Western immunoblot analyses (Fig. 1). This was in accordance with previously presented data [14]. In that previous work unaltered expression of SR Ca²⁺-ATPase and phospholamban after transfection of Ad-NCX could be demonstrated.

View larger version (96K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 Virus-dose-dependent increase in NCX-protein and mRNA levels after 48 h culture time demonstrated by mRNA (upper panel) and Western blot (lower panel), respectively. For comparison, GAPDH mRNA and calsequestrin protein levels have also been analyzed. Note that the quantitative comparison of Na+/Ca2+ exchanger protein levels in transfected and non-transfected cells is problematic because the polyclonal antibody raised against the canine protein presumably does not equally react with the transfected dog exchanger and the endogenous rabbit exchanger. Thus, by comparing densitometric units we may overestimate the degree of Na+/Ca2+ exchanger overexpression. Previously [14], in view of these limitations, we have estimated the degree of overexpression under these conditions as 3.3-fold which is similar to the degree of overexpression observed in end-stage human heart failure.

 
3.2 Effect of the Na⁺/K⁺-ATPase inhibitor ouabain
Fractional shortening (FS) which depends on systolic as well as diastolic properties of the myocytes was analyzed at a stimulation frequency of 1 Hz. Basal FS in the absence of ouabain tended to be lower by 20.7% in NCX-transfected (0.023±0.002, n=17) versus GFP-transfected (0.029±0.003, n=16, P=0.06; Fig. 2) myocytes. This is consistent with previous findings and was shown to result from a reduced SR Ca²⁺ load in NCX overexpressing myocytes [14]. Addition of ouabain caused an increase in FS in both types of myocytes. With low ouabain concentration of up to 2 µmol/l, the increase in FS compared to basal FS was similar in both groups (at 2 µmol/l: +55±15 vs. +55±17%, NCX vs. GFP; n.s.). Increasing the ouabain concentration beyond 2 µmol/l did not provoke a significant further increase in FS in the NCX-group while FS increased markedly at concentrations up to 8 µmol/l in the GFP-group. The maximum inotropic response in the NCX-group was significantly reduced by 50.7% (P<0.05, Figs. 2 and 3).

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Dose-dependence of fractional shortening from ouabain in isotonically contracting myocytes after infection with Ad-GFP and Ad-NCX-GFP, respectively. Na+/Ca2+ exchanger-transfected cells displayed a slightly reduced basal fractional shortening (ns) and a significantly reduced maximum inotropic effect with increasing ouabain concentrations. * P<0.05 vs. 0 µmol/l ouabain, # P<0.05 vs. GFP.

 

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Original recordings of isotonically contracting rabbit ventricular myocytes (a typical experiment in each cell type is shown). Fractional shortening is shown relative to the steady state resting cell length for each ouabain concentration. The dose-dependent effect of ouabain in GFP- (left panel) and NCX-transfected (right panel) myocytes is obvious from an increase in fractional shortening. This effect was blunted in NCX-transfected cells (see Fig. 2).

 
The reduced potency of ouabain in NCX overexpressing myocytes results from increased susceptibility to contracture. The median concentration at which half of the cells developed a contracture and lost their rod-shaped form was 4 µmol/l in the NCX-transfected group and 8 µmol/l in the GFP-transfected control cells (P<0.02; Fig. 4). When analyzing relaxation parameters before contracture, the time from peak shortening to 90% of relaxation (RT90%) was significantly longer (298±39 vs. 185±15 ms, P<0.05) and the maximum relaxation velocity was significantly slowed down (0.15±0.04 vs. 0.27±0.04 µm/s, P<0.05) in the NCX-transfected cells (Fig. 5). Moreover, there was a dose-dependent decrease in diastolic cell length from 137±6 (0 µmol/l) to 130±7 (8 µmol/l) in the GFP-group and from 134±4 (0 µmol/l) to 124±18130±7 (8 µmol/l) in the NCX-group. The decrease in resting cell length was slightly more pronounced in the NCX-group (–7.5% in NCX vs. –5.2% in GFP), however, this was not significant.

View larger version (16K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Bar graphs demonstrating mean diastolic parameters at the concentration before the individual cells went into contracture. * P<0.05.

 
3.3 Effect of the Na⁺-channel activator BDF9198
The effect of BDF9198 on contractile parameters was investigated in NCX- and GFP-transfected cells at a concentration of 1 µmol/l. The results are shown in Fig. 6. There were no significant differences in fractional shortening (0.052±0.005 and 0.055±0.007), RT90% (348±31 and 368±28 ms), or maximum relaxation velocity (0.136±0.048 and 0.163±0.042 µm/s) between GFP- and NCX-transfected rabbit myocytes (Fig. 6).

View larger version (17K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 NCX- (n=27) and GFP- (n=21) overexpressing rabbit myocytes were exposed to the sodium channel modulator BDF9198 (1 µmol/l, 1 Hz). No significant difference was found between fractional shortening (left panel), the time to 90% of relaxation (RT90%, middle panel), and the maximum relaxation velocity (right panel).

 

	4. Discussion

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

 
The present work shows that inhibition of the sarcolemmal Na⁺/K⁺-ATPase by ouabain produced differential effects in rabbit myocytes overexpressing the Na⁺/Ca²⁺ exchanger compared to control cells overexpressing GFP. (1) NCX-transfected cells were much more susceptible to ouabain, i.e. they developed contracture at ouabain concentrations significantly lower than that needed for inducing contracture in the control cells. (2) This was preceded by impaired relaxation which may be indicative of diastolic Ca²⁺ overload. (3) With respect to systolic function, ouabain caused a comparable dose-dependent increase in shortening at low concentrations in both cell types. However, only in the control group fractional shortening further increased with high concentrations of the drug. In the NCX-transfected cells the maximum inotropic response was reduced. (4) In contrast, no such differences were observed after exposure of the cells to the Na⁺ channel modulator BDF9198.

The effect of cardiac glycosides on myocardial function is thought to derive from an inhibition of the sarcolemmal Na⁺/K⁺-pump which is the main Na⁺-eliminatory mechanism of the myocyte. The resulting increase in [Ca²⁺]_i is achieved by the action of the Na⁺/Ca²⁺ exchanger either by shifting its reversal potential to more negative potentials, reducing the outward transport of Ca²⁺ during resting membrane potentials or by increasing Ca²⁺ influx by reverse-mode Na⁺/Ca²⁺ exchange during the action potential when the membrane depolarizes [23]. Both mechanisms lead to an increase in intracellular Ca²⁺ and sarcoplasmic reticulum Ca²⁺ load inducing a positive inotropic response. Only small increases in [Na⁺]_i are sufficient to produce a large positive inotropic effect by influencing the reversal potential of the Na⁺/Ca²⁺ exchanger [10].

Recently, in accordance with our findings in isolated rabbit myocytes, Pieske et al. [19] demonstrated that exposure of failing human muscle strip preparations to ouabain (0.03 µmol/l) caused an increased isometric twitch tension at low stimulation rates. However, upon increases in stimulation rates up to 3 Hz, developed force steeply declined, and diastolic tension largely increased in the presence of ouabain. In contrast, after increasing periods of rest, post rest contractions were largely potentiated in the presence of ouabain (0.03 µmol/l) without changes in diastolic tension [19]. Therefore, conditions of elevated [Na⁺]_i may promote inotropic effects at diastolic periods long enough to allow for sufficient SR Ca²⁺ loading. At faster heart rates combined systolic and diastolic contractile dysfunction may occur. Thus, in the present study at 1 Hz and at low ouabain concentrations, diastolic periods may have been sufficiently long to increase SR Ca²⁺ load and contractility without diastolic dysfunction in the NCX-overexpressing rabbit myocytes. However, induction of [Ca²⁺]_i overload at higher ouabain concentrations may have been too extensive for being sufficiently utilized by the SR resulting in disturbed relaxation and cell contracture.

Our data may also be helpful in understanding discrepancies between recent studies in transgenic mice that overexpress the Na⁺/Ca²⁺ exchanger [24] and rabbit myocytes overexpressing the exchange molecule following adenoviral gene transfer [14,25]. Both models are characterized by sole overexpression of the Na⁺/Ca²⁺ exchanger without any other changes in the expression of the major Ca²⁺ cycling proteins. In the rabbit, our group could demonstrate a reduction in basal fractional shortening in electrically stimulated, isotonically contracting myocytes and a blunted frequency-dependent potentiation of shortening in the NCX-transfected cells that was associated with an impaired SR Ca²⁺ load at higher stimulation frequencies [14]. In addition, we were able to demonstrate a frequency-dependent reduction of the amplitude of Ca²⁺ transients measured with Indo-1 [25]. These findings were indicative of an increased importance of forward mode Na⁺/Ca²⁺ exchange and cellular Ca²⁺ loss in rabbit myocytes that overexpress the exchange protein. The degree of overexpression of Na⁺/Ca²⁺ exchanger achieved in this study may be in the same range observed in end-stage failing human hearts [15].

In contrast to the findings in rabbit myocytes, Terraciano et al. [24] made the observation that the transient inward current elicited by caffeine-induced SR Ca²⁺ release was increased in transgenic mice compared to non-transgenic littermates indicating increased SR Ca²⁺ content. Since no changes in the DHP receptor density were found, the authors concluded that this was a result of increased Ca²⁺ influx by reverse mode Na⁺/Ca²⁺ exchange. The species dependent differences might be related to a differential role of reverse-mode Na⁺/Ca²⁺ exchange due to differences in [Na⁺]_i. In mice, action potential is short, expression of Na⁺/Ca²⁺ exchanger is low and [Na⁺]_i concentration is high [10,26,27]. In particular, the latter condition favors Ca²⁺ entry by reverse-mode Na⁺/Ca²⁺ exchange in wild type mice that may become even more pronounced in transgenic animals overexpressing Na⁺/Ca²⁺ exchanger [26,27]. Of note, changes in [Na⁺]_i homeostasis must not necessarily mean changes in bulk [Na⁺]_i. Inhibition of Na⁺/K⁺-ATPase might result in changes of the submembrane [Na⁺]_i. These submembrane changes of [Na⁺]_i are actually sensed by the Na⁺/Ca²⁺ exchanger and may have pronounced effects on its reversal potential [28].

It should be mentioned that isolated cells may behave differently from multicellular preparations or in vivo conditions regarding contractile properties. This is partly related to the fact that isolated myocytes are devoid of the extracellular matrix, thus, missing elastic elements supporting relaxation. As a result, the optimum stimulation rate for shortening measurements in myocytes is below the physiological heart rate of rabbits. Since NCX activity depends on the relative times spent in diastole and systole and therefore is frequency-dependent, conclusions from experimental findings in isolated myocytes must be drawn carefully with respect to the in vivo situation. However, at higher, more ‘physiological’ heart rates [Na⁺]_i rises which is exaggerated in the failing heart [19], thereby further amplifying impaired relaxation during digitalis treatment [5,8–10].

In view of these limitations we propose that a mechanism that is related to increased Na⁺/Ca²⁺ exchange and reduced function of the Na⁺/K⁺-ATPase could promote diastolic dysfunction in the end-stage failing human heart. In human heart failure, increased expression of the Na⁺/Ca²⁺ exchanger has previously been reported by several authors [1,3,4,15]. Norgaard et al. [17] showed a reduction in ³H-ouabain binding sites in myocardial biopsies from patients with impaired left ventricular function obtained during left heart catheterization suggesting decreased Na⁺/K⁺-ATPase. Consistently, Pieske et al. [19] measuring [Na⁺]_i in muscle strips from end-stage failing human hearts loaded with SBFI demonstrated a significant elevation of [Na⁺]_i at different stimulation rates. At high stimulation rates this was associated with marked diastolic dysfunction [19]. Moreover, Schwinger et al. [18] found a significant isoform specific decrease in the expression of the Na⁺/K⁺-pump and a reduction in the ouabain-binding sites in end-stage failing human hearts from transplant recipients. In contrast to other studies the authors found unchanged NCX protein levels [18]. In the same study [18], the authors investigated the effect of BDF9198 in papillary muscle strips from end-stage failing compared to nonfailing human myocardium using increasing doses of the drug (0.1–10 µmol/l). The maximum inotropic effect was reached at 0.1 µmol/l in failing and 1.0 µmol/l in nonfailing myocardium which demonstrated an enhanced potency of the drug with unaltered effectiveness. Thus, the activity of the Na⁺/K⁺-ATPase seems of major importance for the effect of BDF9198. The lack of difference between NCX- and GFP-transfected rabbit myocytes with respect to the pharmacological effect of BDF9198 (1 µmol/l) observed in the present work may therefore be related to the fact that in both cell types the Na⁺/K⁺-ATPase was functionally intact. It may be speculated that increased Na⁺ entry with BDF9198 is predominantly eliminated by Na⁺/K⁺-ATPase without influencing Na⁺/Ca²⁺ exchange.

In addition, in heart failure other Na⁺-transport systems might well constitute important mechanisms responsible for increased [Na⁺]_i. In this issue, Baartscheer and coworkers [21,29] demonstrate the significance of the Na⁺/H⁺ exchanger as an underlying mechanism of increased [Na⁺]_i in heart failure. Moreover, Despa et al. [20] using a combined aortic insufficiency and constriction heart failure model in the rabbit demonstrated elevation of [Na⁺]_i despite unaltered Na/K-pump characteristics. Most of the excess resting Na⁺ influx in this model occurred through a tetrodotoxin-sensitive pathway suggesting a larger number of Na⁺ channels open in quiescent failing myocytes versus control [20]. Thus, alterations in [Na⁺]_i are likely to be the consequence of multiple systems.

In conclusion, increased expression of Na⁺/Ca²⁺ exchanger could favor the Ca²⁺ efflux as well as the influx mode, or both, depending on the phase of the membrane potential and the transmembrane gradients for Na⁺ and Ca²⁺. The expression and function of the Na⁺/K⁺-ATPase may be of particular importance. When the function of the Na⁺/K⁺-pump is intact, [Na⁺]_i is low. As a consequence, the Na⁺/Ca²⁺ exchanger preferably works in the forward mode and may help as a compensatory mechanism to prevent [Ca²⁺]_i overload and diastolic dysfunction in heart failure. This however may at the same time lead to sarcoplasmic reticulum Ca²⁺ depletion and cellular Ca²⁺ loss followed by systolic contractile dysfunction. Vice versa, when the Na⁺/K⁺-pump is downregulated in end-stage heart failure or when its function is impaired due to digitalis treatment, [Na⁺]_i increases and the reverse mode of Na⁺/Ca²⁺ exchange gets more important. Ca²⁺ influx via reverse mode Na⁺/Ca²⁺ exchange may contribute to SR Ca²⁺ uptake and may compensate for decreased SR Ca²⁺-ATPase expression and function. At the same time, however, the propensity to cellular Ca²⁺ overload and diastolic dysfunction is increased. Therefore, the potential adverse effect of digitalis treatment regarding diastolic dysfunction may be critically exaggerated in patients with congestive heart failure and increased Na⁺/Ca²⁺ exchanger expression and function. Hence, the functional consequences of Na⁺/Ca²⁺ exchanger overexpression in failing myocardium critically depend on the concomitant changes.

Time for primary review 28 days.

	Acknowledgements

 
This work was supported by the Deutsche Forschungsgemeinschaft, grant HA 1233/6-1 to Gerd Hasenfuss and by the Forschungsförderungsprogramm of the Georg-August-Universität Göttingen to Wolfgang Schillinger. We gratefully acknowledge the expert technical assistance of Michael Kothe, Sandra Ott-Gebauer and Jessica Spitalieri. We thank Ken Philipson for kindly supplying the Ad-NCX-GFP vector.

	References

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

 

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