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Na⁺/Ca²⁺ exchanger overexpression impairs frequency- and ouabain-dependent cell shortening in adult rat cardiomyocytes

¹Laboratory of Muscle Research and Molecular Cardiology, Department for Internal Medicine III, University of Cologne, 50924 Cologne; ²Deutsches Herzzentrum der Technischen Universität München, 81675 München; and ³Institute of Medical Statistics, Informatics, and Epidemiology; University of Cologne, 50924 Cologne, Germany

Submitted 3 May 2003 ; accepted in final form 18 May 2004

	ABSTRACT

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
The Na⁺/Ca²⁺ exchanger (NCX) may influence cardiac function depending on its predominant mode of action, forward mode or reverse mode, during the contraction-relaxation cycle. The intracellular Na⁺ concentration ([Na⁺]_i) and the duration of the action potential as well as the level of NCX protein expression regulate the mode of action of NCX. [Na⁺]_i and NCX expression have been reported to be increased in human heart failure. Nevertheless, the consequences of altered NCX expression in heart failure are still a matter of discussion. We aimed to characterize the influence of NCX expression on intracellular Ca²⁺ transport in rat cardiomyocytes by adenoviral-mediated gene transfer. A five- to ninefold (dose dependent) overexpression of NCX protein was achieved after 48 h by somatic gene transfer (Ad.NCX.GFP) versus control (Ad.GFP). NCX activity, determined by Na⁺ gradient-dependent ⁴⁵Ca²⁺-uptake, was significantly increased. The protein expressions of sarco(endo)plasmic reticulum Ca²⁺-ATPase, phospholamban, and calsequestrin were unaffected by NCX overexpression. Fractional shortening (FS) of isolated cardiomyocytes was significantly increased at low stimulation rates in Ad.NCX.GFP. After a step-wise enhancing frequency of stimulation to 3.0 Hz, FS remained unaffected in Ad.GFP cells but declined in Ad.NCX.GFP cells. The positive inotropic effect of the cardiac glycoside ouabain was less effective in Ad.NCX.GFP cells, whereas the positive inotropic effect of -adrenergic stimulation remained unchanged. In conclusion, NCX overexpression results in a reduced cell shortening at higher stimulation frequencies as well as after inhibition of sarcolemmal Na⁺-K⁺-ATPase, i.e., in conditions with enhanced [Na⁺]_i. At low stimulation rates, increased NCX expression enhances both intracellular systolic Ca²⁺ and contraction amplitude.

adenovirus; gene transfer; calcium cycling; sarcoplasmic reticulum; contractility

In human failing myocardium, the regulation of intracellular Ca²⁺ homeostasis is altered (8, 14, 28, 37, 56), which is at least in part due to a reduced activity of sarco(endo)plasmic reticulum Ca²⁺-ATPase (SERCA)2a (14, 25, 29, 56). There is ongoing evidence that NCX protein expression and [Na⁺]_i are increased and the activity of the NCX is altered in human heart failure as well (15, 37, 42, 62). On the one hand, it has been suggested that an increase in the expression of NCX may prevent diastolic dysfunction and intracellular Ca²⁺ overload in human failing myocardium (15); on the other hand, it has been shown that alterations in NCX activity reduce the SR Ca²⁺ load, thus promoting contractile dysfunction of the already failing heart muscle (37).

In transgenic mice chronically overexpressing NCX protein, both the forward and reverse mode of the NCX were stimulated (65). Thus, under appropriate conditions, NCX overexpression may increase contractility. However, in rabbit ventricular myocytes, the gene-mediated acute overexpression of NCX results in depressed cardiac function (52, 53), which may be due to a low [Na⁺]_i, which may favor NCX Ca²⁺ extrusion. In mouse and rat myocardium, [Na⁺]_i is high compared with that in nonfailing human hearts and rabbit myocardium (10, 23, 59, 63, 71; for a review, see also Ref. 6). This may favor NCX activation in the reverse mode and thus improve cardiac contractility. These findings might imply that in failing myocardium, where [Na⁺]_i is high (11, 69, 68), increased NCX expression would exert positive inotropic effects. However, the functional implication of altered NCX expression and function is not yet fully understood.

In rat adult cardiomyocytes, the NCX contributes only 10% to the overall Ca²⁺-transporting capacity under both stimulated conditions (2) and in resting cells (3). Thus overexpression of the NCX by adenoviral gene transfer is likely to reveal functional alterations similar to human failing myocardium (4) and may help the understanding of the functional implication of NCX for Ca²⁺ handling and contraction development.

To elucidate the functional consequences of NCX overexpression and to discriminate its dependence on the type of animal model (e.g., depending on [Na⁺]_i), we report here the consequences of an acute five- to ninefold NCX overexpression via adenoviral gene transfer in rat cardiomyocytes. After NCX overexpression, we performed experiments at increasing stimulation frequencies and Ca²⁺ load after -adrenergic stimulation as well as after inhibition of Na⁺-K⁺-ATPase.

	METHODS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

 
Generation of Adenoviral Recombinants

Wistar rats (Charles River) were held and handled in accordance with standard use protocols and animal welfare regulations. The experimental design was approved by proper authorities and controlled by the Animal Welfare Officer of the University of Cologne.

Generation of a recombinant (E1 deficient) adenovirus (serotype 5) carrying the reporter gene green fluorescent protein (GFP) and the dog NCX (33) was performed with the overall strategy developed by He et al. (16). Briefly, dog NCX cDNA (33) was cloned into the shuttle vector pAdTrack-CMV. The resulting construct was cleaved with the restriction endonuclease PmeI to linearize it. This was then cotransformed with the supercoiled backbone adenoviral vector pAdEasy-1 into Escherichia coli strain BJ5183. Recombinants were selected with kanamycin and screened by restriction endonuclease digestion. The recombinant adenoviral construct was cleaved with PacI to expose its inverted terminal repeats and transfected into the packing cell line, subconfluent HEK-293 cells, by using a modified calcium phosphate coprecipitation method (24). The process of viral production can be directly and conveniently followed in HEK-293 cells by visualization of the GFP reporter, which is incorporated into the viral backbone. After 8–10 days, the virus was harvested and then amplified by infecting increasing numbers of packing cells each time, with a final round using a total of 50 x 145-cm² dishes. After 2–3 days, the resultant virus (Ad.NCX.GFP) was purified by CsCl banding, with the final yield at 3.8 x 10¹² particles/ml. A reporter virus producing GFP alone also under the control of the cytomegalovirus promoter was generated (Ad.GFP) at 1.58 x 10¹² particles/ml. Viral titers were determined to be 2.1 x 10¹⁰ plaque-forming units (pfu)/ml for Ad.NCX.GFP and 4.72 x 10⁹ pfu/ml for Ad.GFP (16).

Preparation and Culture of Adult Ventricular Cardiomyocytes

Single calcium-tolerant ventricular myocytes were isolated from 12- to 16-wk-old male Wistar rats by collagenase 2 digestion (175–200 U/ml, Biochrom) as previously described (19, 40) and cultured in medium 199 (M199) (supplemented with MEM vitamins, MEM nonessential amino acids, 25 mmol/l HEPES, 10 µg/ml insulin, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 100 µg/ml gentamicin) on laminin-precoated dishes (5–10 µg/cm²) at a density of 10⁵ cells/cm² in a humidified atmosphere (5% CO₂) at 37°C. The infection of the myocytes with the adenoviruses was performed 6–8 h after cells were plated in M199. After culture at 37°C for 24 or 48 h, myocytes were viewed using fluorescence microscopy with an excitation wavelength of 485 nm and an emission wavelength of 520 nm to positively identify infected cells. The number of viable rod-shaped cells and the percent cells showing GFP fluorescence were counted. The cells were then used in contraction experiments or frozen for Western blot analysis.

Western Blot Analysis

Myocytes preparations 48 h after infection with Ad.GFP as a control or Ad.NCX.GFP with 1 pfu/cell (Ad.NCX.GFP 1) or 10 pfu/cell (Ad.NCX.GFP 10) were lyzed, pooled, and stored at –80°C. Equal amounts of protein (50 µg total protein) from all samples were separated on SDS-polyacrylamide gels (22) and transferred onto polyvinylidene difluoride (PDVF) transfer membranes (66) with modifications as previously described (31). The efficiencies of the transfers were controlled by gel stainings with Coomassie blue R-250 and by visualization of the transblotted proteins onto PVDF membranes with Ponceau S stains. Nonspecific binding was blocked with Tris-buffered saline (TBS) buffer [10 mmol/l Tris·HCl (pH 7.5) and 150 mmol/l NaCl] that contained 5% nonfat milk. Targeted antigens were probed for 16 h with a monoclonal antibody to the NCX diluted 1:2,000 in TBS containing 1% BSA and 0.1% Tween 20 or with antibodies to SERCA (1:1,250), phospholamban (1:1,000), and calsequestrin (1:1,250), respectively. Antibodies were labeled for 2 h with horseradish peroxidase by anti-mouse or anti-rabbit Ig secondary antibody (1:2,000 dilution, Sigma). Targeted antigens were visualized with an enhanced chemiluminescence assay (Amersham). Specific bands were seen at 70, 120, and 160 kDa with the NCX antibody (33, 34, 62), at 105 kDa with the SERCA antibody, at 28 kDa with the phospholamban antibody, and at 53 kDa with the calsequestrin antibody.

Quantification of Immunoreactive Bands

Band densities were evaluated with the use of an Ultrascan laser densitometer (ImageQuant). Densitometric units of bands obtained with the NCX antibody were added (33, 34, 62). We plotted different amounts of proteins to corresponding densitometric units to check the linearity of the assay before each series of blots. The NCX, phospholamban, and SERCA protein levels were normalized to calsequestrin protein levels as an internal standard. Each individual value represents the mean of two independent determinations. (Note that the quantitative comparison of NCX protein levels in NCX- and GFP-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 rat exchanger. Thus by comparing densitometric units we may overestimate the degree of NCX overexpression.)

NCX Activity

Na⁺ gradient-dependent ⁴⁵Ca²⁺ uptake into cardiomyocyte membranes was determined according to the method of Reeves and Sutko (46). Raw homogenates (50 µg, pooled cell preparations) were loaded with sodium in 200 µl of sodium buffer containing (in mM) 160 NaCl, 20 MOPS, and 1 MgCl₂; pH 7.4. Control homogenates were incubated in potassium buffer containing (in mM) 160 KCl, 1 MgCl₂, and 20 MOPS; pH 7.4. Sodium-driven Ca²⁺ uptake was assayed at 37°C in a buffer containing 160 mM KCl, 1 mM MgCl₂, 20 mM MOPS, and 40 µM ⁴⁵CaCl₂ (0.6–0.8 Ci/mmol). The reaction was terminated by the addition of 3 ml of ice-cold stop buffer containing (in mM) 200 KCl, 5 LaCl₃, 1 EGTA, and 5 MOPS; pH 7.4 at 4°C. The filters (GMF-3, Filtrak; Bärenstein, Germany) were washed twice with 3 ml of 140 mM KCl and 1 mM EGTA and then subjected to scintillation counting. The Na⁺ gradient-dependent Ca²⁺ uptake was determinated in two replicated experiments of eight pooled Ad.NCX.GFP cell preparations and eight pooled Ad.GFP cell preparations. Data are presented as the Na⁺ gradient-dependent uptake of ⁴⁵Ca²⁺ [uptake from membranes preincubated in the Na⁺-containing medium was subtracted from uptake from membranes preincubated in the (Na⁺-free) KCl medium]. For the calculation of maximal Ca²⁺ uptake and time to half-maximal Ca²⁺ uptake, nonlinear regression analyses were performed. Regression analyses were performed using the computer software GraphPad Prism (GraphPad Software; San Diego, CA).

Myocyte Contraction

Cell shortening of Ad.GFP- or Ad.NCX.GFP-infected rat ventricular cardiomyocytes was measured with an electrooptical monitoring system in a single cell investigation system (Scientific Instruments; Heidelberg, Germany) in a temperature-controlled cuvette (32°C) at a constant medium flow rate of 0.5 ml/min and under continuous electric field stimulation. Ca²⁺ transients were simultaneously recorded with the fluorescent Ca²⁺ indicator fura-2 using a dual-wavelength fluorometer. Cells were loaded by incubation with fura-2 AM for 20 min at 32°C. The dye was excited at 340- and 380-nm wavelengths alternately, and the emission was measured at 530 nm. The relative Ca²⁺ signal was calculated from the 340-to-380-nm ratio. Ca²⁺-containing Tyrode solution (1.8 mmol/l) was used as the medium. The cardiomyocytes were paced by an external stimulation of 50 V with ascending frequencies from 0.25 to 3.0 Hz (15–180 beats/min) or under a constant frequency of 0.5 Hz for inotropic interventions. Fractional shortening is the shortening amplitude as a percentage of the resting cell length.

Investigation of NCX Phosphorylation Status

The phosphorylation status of NCX after protein kinase A (PKA) stimulation was investigated in Western blots with antibodies that react specifically with phosphorylated serine (as a free amino acid or phosphoserine-containing proteins) and with the "backphosphorylation technique" also. The basic difference between the two phosphorylation experiments was the mode of detection of phosphorylation (serine residues or ³²P incorporation). The in vitro phosphorylations were performed as described recently (57). In brief, myocyte homogenates were phosphorylated by PKA in the following basic reaction mixture containing (in mmol/l): 40 histidine/HCl, 0.01 cantharidin, 100 NaCl, 10 MgCl₂, 15 NaF, and 1 EGTA, with 0.1 % Triton X-100, 100 µg BSA, and 0.5 ng of the catalytic subunit of PKA (PKA-CS) reconstituted in 6% dithiothreitol and 43 µg protein in a final volume of 50 µl; pH 6.8. Cantharidin was added to inhibit protein phosphatases. Cantharidin (10 µM) completely inhibits type 1 and type 2A phosphatases (32). The samples were preincubated for 2 min at 30°C. For the backphosphorylation technique, the reaction was initiated by adding 10 µl [-³²P]ATP (50 µmol/l, 2.5 x 10⁶ counts/min) and allowed to proceed for 10 min. For termination, 25 µl of an ice-cold stop solution containing 1% SDS, 5% mercaptoethanol, 1 mM EDTA, 10 mM Tris·HCl (pH 8), and 20 µl Laemmli buffer (20) were used. The samples were immediately boiled in a water bath for 5 min. Electrophoretic separation was performed in duplicate to 4–12% urea-SDS-PAGE. Autoradiography using X-ray-sensitive film and intensifying screens permitted the detection of ³²P-labeled proteins on the gels. The band densities of NCX were evaluated by densitometric scanning of the whole gel using a computerized imaging system (PDI). ³²P-labeled NCX bands were normalized against total protein recovered from cell membrane preparations. The NCX bands were identified with Western blots after the backphosphorylation on the same gels. For the other mode of detection of phosphorylation (serine residues), contrary to the backphosphorylation, the reaction was started with 10 µl ATP (50 µmol/l) and likewise only 6% dithiothreitol (in place of PKA) was added in the control assays (basal Ad.GFP and basal Ad.NCX.GFP values). Reactions were immediately terminated by boiling in Laemmli buffer for 5 min, and samples were then subjected to SDS-PAGE on a 4–12% gel. For the identification of the NCX band, the samples were run in duplicate on the gels in a way that the resulting blots can be cut at the lane of a prestained molecular marker in two equal halves having same samples on each. One part was used for the anti-NCX antibody, and the other one was used for the detection of serine residues.

Materials

High-grade salts were purchased from Merck (Darmstadt, Germany); MOPS and PKA-CS (bovine heart) were from Sigma (St. Louis, MO). Acrylamide/bisacrylamide (30%) was from Bio-Rad (Hercules, CA). Fura-2 AM was obtained from Molecular Probes (Eugene, OR). The primary antibody used for SERCA2a detection was mouse monoclonal IgG₁ antibody against canine SERCA2a protein (ABR, Affinity Bioreagents; Golden, CO). The anti-phospholamban antibody was mouse monoclonal IgG antibody against canine phospholamban purchased from Upstate Biotechnology (Lake Placid, NY.). The antibody against calsequestrin was polyclonal rabbit antibody against canine calsequestrin from SWant (Bellizona, Switzerland). The antibody against the NCX was polyclonal rabbit anti-NCX antibody from SWant. The monoclonal anti-phosphoserine (mouse) and the secondary antibodies were either monoclonal sheep anti-mouse IgG peroxidase conjugated or goat anti-rabbit IgG conjugated with peroxidase from Sigma Immunochemicals.

Statistical Analysis

Sample distributions of continuous variables are summarized as means ± SE; n refers to the number of studied cells in contraction experiments. For the experiments with viral load and sarcolemmal Ca²⁺ uptake, myocytes in a given preparation were pooled, so that n values refer to the number of preparations used. Comparisons of mean values for protein expression normalized to calsequestrin were performed with the use of one-way ANOVA (three groups). If the main effect was significant at level 5%, then pairwise contrasts were tested (correction of contrast P values were not necessary). Repeated-measures ANOVA with covariance type AR(1) was applied to evaluate group (Ad.NCX.GFP, Ad.GFP) differences in the mean values of several variables (Ca²⁺ uptake, fractional shortening etc., i.e., Figs. 3 and 5–7). If main or interaction effects were significant at level 5%, then pairwise constrasts between and within groups were tested (contrast P values were Bonferroni corrected). Differences within single pairs of means (e.g., the control vs. overexpressing group at the maximal level of response, half-maximal uptake rate, and EC₅₀ values) were evaluated using the unpaired t-test (no multiplicity correction). Use of a variance-stabilizing transformation (logarithm) did not improve ANOVA assumptions; thus, relying on the robustness of ANOVA results, all variables were analyzed on their original scale. The statistical analyses were performed with the software Prism 4 and SPSS 11.0 for Windows.

View larger version (14K): [in this window] [in a new window]   Fig. 3. NCX activity in rat cardiomyocytes after GFP or Ad.NCX.GFP adenoviral gene transfer. The time dependence of the Na+ gradient-dependent Ca2+ uptake was determined in homogenized cardiomyocytes. 45Ca2+ uptake into homogenate vesicles from rat cardiomyocytes infected with Ad.NCX.GFP (n = 8 preparations) is compared with that in membranes from rat cardiomyocytes infected with Ad.GFP (n = 8 preparations). The Na+ gradient-dependent Ca2+ uptake was significantly higher in NCX-overexpressing cardiomyocytes. Until the first 60 s there were significant differences between both groups (P < 0.001). *P < 0.05 vs. basal; #P < 0.01 vs. Ad.GFP; ##P < 0.001 vs. Ad.GFP obtained by 2-way ANOVA (Bonferroni-corrected post hoc comparisons).

View larger version (18K): [in this window] [in a new window]   Fig. 5. Frequency-dependent alterations of fractional shortening (A), relaxation (B), and amplitude of variation of the fura-2 ratio (C) measured in isolated cardiomyocytes after infection with Ad.GFP and Ad.NCX.GFP. A: fractional shortening was increased at low but decreased at higher stimulation frequencies in parallel to relaxation time. *P < 0.05 vs. basal; #P < 0.05 vs. Ad.GFP. B: relaxation time was significantly shorter (P = 0.015) in Ad.NCX.GFP cells at higher stimulation frequencies compared with Ad.GFP cells. C: amplitude of the systolic change in the fura-2 ratio at 0.5–3 Hz in GFP- compared with NCX-infected myocytes. Although the ratio at 0.25 Hz was significantly higher in NCX-infected cells suggesting higher intracellular Ca2+ levels, the opposite was true for all higher stimulation frequencies.

View larger version (16K): [in this window] [in a new window]   Fig. 7. Influence of inotropic stimulation with extracellular Ca2+ after adenoviral overexpression of the NCX. The increase in fractional shortening of Ad.NCX.GFP-transfected cardiomyocytes (n = 8 cells) superfused with increasing Ca2+ concentration from 2 to 7 mmol/l. was even greater than GFP-transfected cardiomyocytes (n = 9 cells). A stimulation rate at 0.5 Hz was used, and shortening is expressed relative to diastolic length (fractional shortening in %). *P < 0.05 vs. basal; #P < 0.05 vs. Ad.GFP obtained by 2-way ANOVA (Bonferroni-corrected post hoc comparisons).

	RESULTS

TOP ABSTRACT METHODS RESULTS DISCUSSION GRANTS REFERENCES

Infection of adult rat cardiomyocytes with the Ad.GFP virus resulted in a robust expression of the reporter gene GFP visible by green fluorescence with fluorescence microscopy. Adenoviral gene transfer of Ad.NCX.GFP was both dose dependent and time dependent. Twenty-four hours after the infection with Ad.NCX.GFP, 69.3 ± 3.5% of rod-shaped myocytes contained detectable GFP; by 48 h, this percentage rose to 93.2 ± 3.3% and at 72 h the respective value was 96.4 ± 3.7% at a multiplicity of infection (MOI) of 10 pfu/cell.

The transfection efficiency was 78% with a MOI of 1 pfu/cell and over 90% with 10 pfu/cell at 48 h after transfection. Infection of the cells with 1 and 10 pfu Ad.NCX.GFP/cell resulted in a dose-dependent overexpression of NCX protein as determined by immunoblotting 48 h after transfection (see Fig. 1A), as indicated by a MOI-dependent increase in immunoreactive bands at 120 and 160 kDa corresponding to NCX protein (33, 34, 62). Also, when protein levels were normalized to calsequestrin, a MOI-dependent expression was detected (Fig. 2). SERCA2a, phospholamban, and calsequestrin, involved in SR Ca²⁺ uptake and storage, remained unaffected by the somatic gene transfer of Ad.NCX.GFP (see Fig. 1B).

View larger version (85K): [in this window] [in a new window]   Fig. 1. Expression of Ca2+-regulating proteins in adult rat cardiomyocytes after adenoviral gene transfer of an adenovirus carrying the dog Na+/Ca2+ exchanger (NCX) under the control of a cytomegalovirus promoter (Ad.NCX.GFP). A: protein expression of NCX in control cardiomyocytes infected with an adenovirus carrying the reporter gene green fluorescent protein (GFP; Ad.GFP) compared with cardiomyocytes infected with increasing virus doses of Ad.NCX.GFP at a multiplicity of infection (MOI) of 1 plaque-forming units (pfu)/cell (Ad.NCX.GFP 1) or 10 active virus particles per cell (10 pfu/cell; Ad.NCX.GFP 10). The characteristic bands at 70, 120, and 160 kDa of the NCX with increasing intensity are demonstrated by Western immunoblot analysis. B: protein levels of sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)2a (at 105 kDa) and phospholamban (PLB; at 28 kDa) were not affected by adenoviral gene transfer of NCX as shown in these representative Western blots. The density of the calsequestrin band (at 53 kDa) served as an internal standard. The size of molecular mass markers is indicated on the left (calculated kDa in brackets).

View larger version (14K): [in this window] [in a new window]   Fig. 2. Plots showing results of Western blot analysis of NCX, SERCA2a, and PLB normalized to calsequestrin protein levels from myocytes preparation 48 h after infection with Ad.GFP as a control (open squares) or Ad.NCX.GFP with 1 pfu/cell (shaded squares) or 10 pfu/cell (solid squares); therefore, ratio has no unit. Each dotted line represents the mean of 12 cell membrane preparations. Western immunoblot analysis and normalization to calsequestrin showed significantly increased NCX protein levels in Ad.NCX.GFP-transfected myocytes with increasing virus doses. Protein levels of SERCA2a and PLB also normalized to calsequestrin were unchanged. Data for these plots are summarized in Table 1.

Na⁺ Gradient-Dependent Ca²⁺ Uptake

To study whether NCX protein overexpression influences NCX activity, the Na⁺ gradient-dependent Ca²⁺ uptake into rat myocardial vesicles was measured. The Na⁺-driven Ca²⁺ uptake increased in a time-dependent manner from 5 to 120 s in Ad.GFP and Ad.NCX.GFP cells. The maximal NCX rate of Na⁺-driven Ca²⁺ uptake was significantly higher in Ad.NCX.GFP-infected cardiomyocytes (maximal uptake rate: 1.89 ± 0.31 pmol Ca²⁺/µg protein) compared with Ad.GFP-infected controls (maximal uptake rate: 0.73 ± 0.11 pmol Ca²⁺/µg protein). Moreover, Na⁺-dependent Ca²⁺ uptake was significantly faster in Ad.NCX.GFP-infected cardiomyocytes as indicated by a shortening in time to half-maximal uptake rate (Ad.NCX.GFP: 8.43 ± 6.0 s) compared with Ad.GFP controls (23.33 ± 5.1 s). The Na⁺-dependent Ca²⁺ uptake was increased about threefold in the Ad.NCX.GFP cell preparations (see Fig. 3). The comparison of the whole curves between groups (two-way ANOVA) yielded the following: [main effects] group, P = 3.1 x 10^–7; time, P = 2.7 x 10^–9; and [interaction] group x time, P = 1.4 x 10^–5. Thus NCX overexpression (5- to 9-fold) was followed by an increase in Na⁺ gradient-dependent Ca²⁺ uptake, i.e., by a threefold increase.

Contraction

Frequency-dependent cell shortening. To investigate the role of increased NCX activity on cardiac function, the frequency-dependent cell shortening was measured in isolated adult rat cardiomyocytes overexpressing either the GFP or GFP and NCX protein (extracellular Ca²⁺ concentration of 1.8 mM). Figure 4 gives original shortening traces and Ca²⁺ transients (fura-2) at stimulation frequencies of 0.25 and 3 Hz. Figure 5 and Table 2 summarize the results. The fractional shortening at very low stimulation rates (0.25 Hz) was significantly higher in Ad.NCX.GFP cardiomyocytes (n = 38 cells) compared with Ad.GFP cardiomyocytes (n = 30 cells; Table 2). This holds also true for the amplitude of the fura-2 transient (60, 61). At 0.5 Hz, fractional cell shortening and the fura-2 amplitude were similar in NCX- and GFP-overexpressing cells. At higher stimulation rates, the fura-2 transient (see Fig. 4) and fractional cell shortening were significantly depressed in NCX-overexpressing cells infected with Ad.NCX.GFP compared with Ad.GFP (see Fig. 5A and Table 2). A frequency-dependent decrease in diastolic cell length was observed in both NCX- and GFP-overexpressing cells with no difference between both groups at all frequencies measured (two-way ANOVA yielded the following: [main effects] group, P = 0.07; frequency, P = 3.5 x 10^–6; and [interaction] group x frequency, P = 1.1 x 10^–5). Shortening velocity was similar at all stimulation frequencies, whereas relaxation velocity was significantly decreased in the Ad.NCX.GFP group. Relaxation time as measured by the time needed for 50% decrease of maximal cell shortening frequency dependently decreased in both Ad.GFP and Ad.NCX.GFP cells. Relaxation time was significantly shorter in Ad.NCX.GFP cells at higher stimulation frequencies (2-way ANOVA yielded the following: [main effects] group, P = 0.011; frequency, P = 3.2 x 10^–9; and [interaction] group x frequency, P = 0.54) compared with Ad.GFP cells (Fig. 5B). However, cell shortening was reduced in Ad.NCX.GFP cells at high stimulation rates as well. Thus overexpression of NCX protein on adult rat cardiomyocytes may result in a shortening of cardiac relaxation at higher stimulation frequencies and may be accompanied by a reduced SR Ca²⁺ load. Figure 5C depicts fura-2 ratios from 0.5 to 3 Hz comparing GFP- and NCX-infected myocytes. Although the ratio at 0.25 Hz was significantly higher in NCX-infected cells, suggesting higher intracellular Ca²⁺ levels, the opposite was true for all higher stimulation frequencies. Comparison of the whole curves between groups yielded the following: [main effects] group, P = 0.32; frequency P = 6.3 x 10^–8; and [interaction] group x frequency, P = 2.8 x 10^–9.

View larger version (16K): [in this window] [in a new window]   Fig. 4. Contraction amplitude and Ca2+ transients in adult rat cardiomyocytes after adenoviral overexpression of the NCX. Representative tracings of the contraction cycle (A) and Ca2+ transient (B) of single rat cardiomyocytes after stimulation rates with 0.25 and 3 Hz are shown. A: cardiomyocyte transfected with a control adenovirus carrying GFP (Ad.GFP) is compared with an Ad.NCX.GFP-transfected cardiomyocyte (Ad.NCX.GFP). The contraction amplitude is presented as changes of the cell length during the contraction cycle expressed as micrometers. The Ca2+ transients (B) are measured with the Ca2+ indicator fura-2 AM at the characteristic wavelengths of 340 and 380 nm and expressed as a ratio of the light signal (R340/380).

To investigate whether the NCX in a situation of enhanced SR Ca²⁺ uptake may still compete with SERCA2a, fractional shortening of Ad.GFP- and Ad.NCX.GFP-infected cardiomyocytes was measured at increasing concentrations of isoprenaline, which is known to phosphorylate phospholamban and thereby improve SR Ca²⁺ uptake by SERCA.

The positive inotropic effect after cumulative isoprenaline application (1–100 nM) was similar in NCX- and GFP-overexpressing rat cardiomyocytes [fraction shortening: basal vs. plus isoprenaline (100 nM); Ad.GFP: 4.69 ± 0.21% vs. 10.57 ± 0.68%; and Ad.NCX.GFP: 4.51 ± 0.49 vs. 9.85 ± 0.30, P > 0.05; Fig. 6A ]. The same holds true for the EC₅₀ values for the concentration-dependent effects of isoprenaline: 2.20 ± 0.62 nM for the NCX-overexpressing group and 2.36 ± 0.66 nM for the GFP-overexpressing group (P = 0.86). Two-way ANOVA yielded the following: [main effects] group, P = 0.27; isoprenaline, P = 5.5 x 10^–18; and [interaction] group x isoprenaline, P = 0.69.

View larger version (18K): [in this window] [in a new window]   Fig. 6. A: contraction amplitudes of rat myocytes after Ad.NCX.GFP and Ad.GFP infection in response to increasing concentrations of isoprenaline. The positive inotropic effect after cumulative isoprenaline application (1–100 nM) was similar in NCX-overexpressing (n = 11 cells) and GFP-overexpressing (n = 10 cells) rat cardiomyocytes. *P < 0.05 vs. basal. B: contraction amplitudes of rat myocytes after Ad.NCX.GFP and Ad.GFP infection in response to increasing ouabain concentrations in the perfusion solution. In both GFP-transfected (n = 8 cells) and NCX.GFP-transfected cells (n = 8 cells), fractional cell shortening was significantly increased with elevated ouabain concentrations. The maximal positive inotropic effect of ouabain (50 µM) was significantly decreased in isolated cardiomyocytes overexpressing NCX and GFP compared with cells expressing only GFP. *P < 0.05 vs. basal; #P < 0.05 vs. Ad.GFP.

Ouabain-Dependent Cell Shortening

It has been demonstrated that the NCX is functionally linked to sarcolemmal Na⁺-K⁺-ATPase (9, 49). To investigate the influence of NCX overexpression on NCX-Na⁺/K⁺ pump interaction, fractional cell shortening was measured at a stimulation frequency of 0.5 Hz and an extracellular Ca²⁺ concentration of 1.8 mM (i.e., under conditions of effective cell shortening) at increasing ouabain concentrations (10–100 µM). The results are presented in Fig. 6B. In both GFP- and NCX-transfected cells, fractional cell shortening was significantly increased at increasing ouabain concentrations (two-way ANOVA yielded the following: [main effects] group, P = 0.049; ouabain, P = 1.7 x 10^–23; and [interaction] group x ouabain: P = 2.8 x 10^–5). These ANOVA results (P values < 0.05 for group and for the [interaction] group x ouabain concentration) indicate that the overall contractile response to ouabain was lower in the Ad.NCX.GFP group. The maximal positive inotropic effect of ouabain (50 µM), however, was significantly decreased in isolated cardiomyocytes overexpressing NCX protein (+76.1 ± 8.2% of basal cell shortening, n = 8 cells) compared with cells overexpressing GFP (+136.3 ± 22.3% of basal cell shortening, n = 8 cells). The ouabain sensitivity as measured by the EC₅₀ concentration of ouabain was similar in NCX-overexpressing cells (14.6 ± 1.8 µM) and GFP-overexpressing cells (17.8 ± 2.4 µM, P > 0.05).

Ca²⁺-Induced Positive Inotropic Effect

To investigate whether an increase in NCX protein may help to avoid intracellular Ca²⁺ overload, cell shortening was measured in Ad.NCX- and Ad.NCX.GFP cells at increasing extracellular Ca²⁺ concentrations (2–7 mM) at a stimulation frequency of 0.5 Hz. Inotropic stimulation with increasing Ca²⁺ concentrations from 2 to 7 mM elicited a significantly increased shortening in NCX-overexpressing cardiomyocytes (n = 8 cells) compared with GFP-expressing controls (n = 9 cells, maximal positive inotropic effect: Ad.GFP, +3.1 ± 1.2%; Ad.NCX.GFP, +5.9 ± 1.9%; Fig. 7). Diastolic cell length was significantly decreased in both groups at increasing extracellular Ca²⁺ to a similar extent (7 mM Ca²⁺, Ad.GFP: 91.6 ± 1.5%, Ad.NCX.GFP: 89.3 ± 2.7% of basal diastolic cell length, P = 0.55). For peak shortening, two-way ANOVA yielded the following: [main effects] group, P = 0.044; Ca²⁺: 2.8 x 10^–10; and [interaction] group x Ca²⁺: P = 0.011.

	DISCUSSION

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[Na⁺]_i and hence activation of the NCX are largely species dependent. In mouse (63, 71) and rat (10, 23, 59) myocardium, [Na⁺]_i is high compared with rabbit or human myocardium (10), which favors the reverse mode of action of the NCX, i.e., Na⁺-driven Ca²⁺ inward movement. The reverse mode of the NCX predominates over the direct mode at low stimulation frequencies [as could be observed in cat ventricles (67)]. Usually the NCX operates in the forward mode, in which Na⁺ enters the cell and Ca²⁺ is extruded, playing a central role in the control of muscle relaxation and diastolic calcium (7, 2). By these actions, the NCX indirectly regulates Ca²⁺ stored in the SR. In the diseased state, e.g., human heart failure, both [Na⁺]_i (39) and NCX (62) protein expression may be increased. These changes may influence Ca²⁺ handling and the regulation of myocardial contraction and relaxation. To study the role of myocardial NCX on Ca²⁺ handling and contraction development, we used adenoviral gene transfer to overexpress NCX protein by five- to ninefold in adult rat cardiomyocytes. Because the NCX contributes only a little (10%) to the overall Ca²⁺-transporting capacity in the rat, this species was studied to investigate changes in Ca²⁺ handling during excitation-contraction coupling associated with NCX overexpression. Besides this enhancement in NCX protein, there was no change in protein levels of phospholamban, calsequestrin, or SERCA2a, i.e., no compensatory changes of main Ca²⁺-regulatory proteins could be detected.

In these NCX-overexpressing rat cardiomyocytes, we studied the frequency-dependent cell shortening as well as the effect of inotropic stimulation. Cell shortening at a low stimulation rate of 0.25 Hz was significantly increased in Ad.NCX.GFP myocytes compared with control myocytes. This corresponds well to the observed systolic variation of the fura-2 ratio at this stimulation frequency in NCX-overexpressing myocytes. One contributing mechanism could be the action of NCX in the reverse mode in the early phase of depolarization, i.e., during the increase in [Na⁺]_i. This may be further supported by the high intracellular Na⁺ levels in rat (10, 23, 59) and mouse (63, 71) myocytes. In transgenic mice overexpressing NCX, the SR Ca²⁺ content was increased (64, 65). In contrast, in rabbit ventricular myocytes (i.e., a species with low [Na⁺]_i), cell shortening was depressed after adenoviral gene transfer of the NCX (53).

The mode of action of the NCX may be affected after higher stimulation frequencies, e.g., with increased stimulation frequency, the Ca²⁺ transport in the forward mode of the NCX decreases in rabbit myocytes (1), and this change may be species dependent as well. After a further increase in the stimulation rate in rat myocytes overexpressing the NCX, fractional shortening was diminished (present study). This may result from a reduced SR Ca²⁺ load, e.g., after a shortening of the time of the NCX acting in the reverse mode (59). In addition, more NCX activity may be directed to Ca²⁺ efflux leading to a lower SR Ca²⁺ load. Thus SR Ca²⁺ load may not be sufficient for further increase in contraction. This goes in line with recent findings in human failing myocardium with enhanced NCX protein expression (15). After an increased stimulation rate, force of contraction declines in human failing myocardium (15, 30, 55). Hasenfuss et al. (14) demonstrated that disturbed diastolic function occurs in human myocardium with decreased SERCA, i.e., in a condition with reduced SR Ca²⁺ uptake. In contrast, increased expression of NCX protein was associated with preserved diastolic function in diseased human myocardium (62) possibly after an more effective Ca²⁺ extrusion mechanism (forward mode).

The frequency of stimulation may help to discriminate between preserved cardiac function and compromised cardiac function. Possibly an enhanced NCX activity (e.g., in human failing myocardium) may preserve relaxation at moderately increased heart rates, but this effect may be influenced by additional factors as well (e.g., SERCA activity, etc.). After a further increase in the stimulation rate, both systolic and diastolic dysfunction may occur, depending on the basal intracellular Na⁺ load, also. Corresponding to that, Pieske et al. (38) found, in failing human muscle strips, ouabain, i.e., after an increase in [Na⁺]_i, caused inotropic effects at low stimulation frequencies only. Thus enhanced [Na⁺]_i may initiate positive inotropic effects only at frequencies in which during the diastolic period the SR is filled up with no concomitant increase in diastolic intracellular Ca²⁺ concentration ([Ca²⁺]_i) levels.

To study the impact of [Na⁺]_i on NCX function further, cell shortening was enhanced by inhibition of Na⁺-K⁺-ATPase. Reuter et al. (48) showed that the NCX is a prerequisite for the effect of cardiac glycosides on [Ca²⁺]_i and contractility. In the present study, ouabain was more effective in control compared with Ad.NCX.GFP cardiomyocytes to enhance cell shortening. At low stimulation rates (0.5 Hz), and at low ouabain concentrations (up to 10 µM), there were no differences between the groups. In the rat (already high [Na⁺]_i), a further increase in [Na⁺]_i may not add substantial stimulation of the NCX in the reverse mode. Similarly, increasing the rate was not effective at all in any of the groups in rat myocytes (and had just the opposite effect in the overexpression group). After treatment with ouabain, which favors the reverse mode of the NCX, both the Ca²⁺ influx and SR Ca²⁺ load were enhanced (46). However, the probability of Ca²⁺ overload, especially in a situation with increased expression of the NCX, is enhanced. Thus low concentrations of cardiac glycosides may be more favorable in treatment of patients with compromised cardiac function and increased NCX expression (60) as has been demonstrated recently (44).

Terracciano and co-workers (65) reported increased SR Ca²⁺ content in transgenic mice overexpressing the NCX. This may result from increased Ca²⁺ influx via activation of the exchanger in the reverse mode. In the present study, increased contractility was observed in Ad.NCX.GFP cells after inotropic stimulation after enhanced extracellular Ca²⁺ levels. Under these conditions, cell shortening was significantly increased in Ad.NCX.GFP cardiac myocytes. In contrast, there was no difference in the effect of the -agonist isoprenaline on parameters of contractility between the two groups. The increase in cell contraction development via -agonist stimulation is accompanied by a more pronounced elevation in [Ca²⁺]_i compared with inotropic effects after elevation of Ca²⁺ concentration extracellularly (e.g., due to a Ca²⁺ desensitizing effect of PKA-dependent phosphorylation on the contractile apparatus) (13). Because no changes on contraction development after -stimulation have been observed (in Ad.NCX.GFP-infected myocytes vs. controls), NCX expression may not effect contraction development due to differences in the phosphorylation status of the NCX.

In diseased human myocardium, the Na⁺-K⁺-ATPase activity, its protein abundance, and isoform composition are altered (58), which has also been shown to be of functional relevance (11, 55). In human failing myocardium, the Na⁺ channel modulator 4-[3-(1-diphenylmethyl-azetidin-3-oxy)-2-hydroxy-propoxy]-1H-indol-2-carbonitril (BDF-9148) has been shown to change the force-frequency relationship even in failing human myocardium to a positive one (12, 55). These effects were observed only when BDF-9148 was used in low concentrations that showed no inotropic effects, i.e., concentrations that exerted changes in [Na⁺]_i without altering [Ca²⁺]_i. When high concentrations of BDF-9148 (3 µM) (i.e., with significant changes in [Ca²⁺]_i) were used, the force-frequency relationship was negative. The NCX activity may be affected by changes in [Na⁺]_i and [Ca²⁺]_i in a very sensitive manner. The differences in the force-frequency relationship in human failing and nonfailing tissue may also result from differences in [Na⁺]_i influencing NCX activity and thereby affecting the SR Ca²⁺ load. Pieske and co-workers (38) elegantly demonstrated enhanced [Na⁺]_i in failing human myocardium. These changes may have an impact on both Na⁺ and Ca²⁺ homeostasis. However, this may be secondary to changes in Na⁺-K⁺-ATPase and/or the NCX expression level or activation pattern. Therefore, NCX expression may influence parameters of contraction and relaxation, depending on membrane potential during the course of an action potential, as well as the gradient for Na⁺ and Ca²⁺, e.g., BDF-9148 was effective only at low concentrations to influence force-frequency behavior in failing human myocardium. Thus these changes in diseased human myocardium may result from increased [Na⁺]_i (38), from reduced Na⁺/K⁺ ATPase expression (58), from increased NCX expression (15, 38), or from a combination of these changes observed in failing human myocardium.

In conclusion, the consequences of NCX overexpression on parameters of contraction and relaxation largely depend on concomitant changes in [Na⁺]_i, [Ca²⁺]_i, action potential, and Na⁺-K⁺-ATPase and the studied species (43, 52, 72).

NCX overexpression results in a blunted cell shortening at higher stimulation frequencies and after inhibition of sarcolemmal Na⁺-K⁺-ATPase, i.e., in conditions with already elevated [Na⁺]_i. These situations demonstrate inhibition of the forward mode and activation of the reverse mode of the NCX. Thus increased NCX expression in cardiac tissue may deteriorate rather than improve cardiac function especially under situations with already increased [Na⁺]_i.

Limitations of the Study

Intracellular Ca²⁺ handling is a very complex mechanism not only affected by NCX, SERCA2a, and L-type Ca²⁺ channel expression or function. In addition, changes in Ca²⁺ handling may not only influence contractile function but also regulatory processes (e.g., myocardial hypertrophy). The findings observed in isolated cardiomyocytes or cell membrane preparations may give only indirect information on the physiological or pathophysiological changes present in vivo. It is of importance that there is yet no clear and sufficient answer to the question of why contraction or Ca²⁺ handling is altered. The present findings may give additional insight on the interaction between NCX and Ca²⁺ regulation. However, these mechanisms may be species and disease dependent.

	GRANTS

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Experimental work was supported by the Marga and Walter Boll Foundation (Marga und Walter Boll Stiftung, Köln, Germany).

	ACKNOWLEDGMENTS

 
Our special thanks go to Katja Rösler for excellent technical assistance.

This work contains part of the doctoral thesis of P. Mackenstein.

	FOOTNOTES

 

Address for reprint requests and other correspondence: R. H. G. Schwinger, Laboratory of Muscle Research and Molecular Cardiology, Dept. of Internal Medicine III, Univ. of Cologne, Joseph-Stelzmann Strasse 9, 50924 Cologne, Germany (E-mail: Robert.Schwinger{at}medizin.Uni-Koeln.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^* B. Bölck and G. Münch contributed equally to this work.

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