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(Circulation. 1996;94:992-1002.)
© 1996 American Heart Association, Inc.

Articles

Evidence for Functional Relevance of an Enhanced Expression of the Na⁺-Ca²⁺ Exchanger in Failing Human Myocardium

Markus Flesch, MD; Robert H.G. Schwinger, MD; Frank Schiffer; Konrad Frank; Michael Sudkamp, MD; Ferdinand Kuhn-Regnier, MD; Georg Arnold, MD; Michael Bohm, MD

Klinik III fur Innere Medizin (M.F., R.H.G.S., F.S., K.F., M.B.), Klinik fur Herz und Gefaßchirurgie (M.S., F.K.-R.), and Institut fur Pathologie der Universitat zu Koln (G.A.), Koln, Germany.

Correspondence to Prof Dr med M. Bohm, Klinik III fur Innere Medizin der Universitat zu Koln, Joseph Stelzmann Str 9, 50924 Koln, FRG.

	Abstract

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Background The present study aimed at investigating the expression of the Na⁺-Ca²⁺ exchanger and its functional role in human failing myocardium.

Methods and Results Na⁺-Ca²⁺ exchanger mRNA and protein levels were examined in nonfailing (NF, n=8) and failing human myocardium (New York Heart Association functional class IV) with idiopathic dilated cardiomyopathy (DCM, n=8) or ischemic heart disease (ICM, n=6). The inotropic effect of the Na⁺ channel activator BDF 9148 was determined in electrically driven left ventricular papillary muscle strip preparations (1 Hz, 37°C) from nonfailing (n=8) and failing (n=8) human hearts. Na⁺-Ca²⁺ exchanger mRNA levels were significantly increased, by 79% (P<.001) in DCM and by 58% (P<.01) in ICM compared with NF; protein levels increased by 36% (P<.001) and by 20% (P<.05), respectively. BDF 9148 increased the force of contraction concentration dependently, with a similar maximal effect in NYHA class IV and NF, but was more potent in NYHA class IV as demonstrated by a significantly smaller (P<.01) EC₅₀ value (NYHA class IV, 0.18 [0.16 to 0.22] µmol/L; NF, 1.65 [1.3 to 3.0] µmol/L). In NYHA class IV, BDF 9148 (0.1 µmol/L) restored the positive force-frequency relationship and reduced the frequency-dependent increase in diastolic tension in relation to force of contraction.

Conclusions The increased expression of the Na⁺-Ca²⁺ exchanger is a possible explanation for the increased inotropic potency of the Na⁺ channel activator BDF 9148 in failing human myocardium. The increase in exchanger molecules could be of functional relevance for the modulation of cardiac contractility by agents that increase the intracellular Na⁺ concentration. Enhancement of Na⁺-Ca²⁺ exchanger activity might be a powerful mechanism for increasing cardiac contractility in chronic heart failure.

Key Words: heart failure • calcium channels • inotropic agents • sodium • excitation

	Introduction

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The cardiac sarcolemmal Na⁺-Ca²⁺ exchanger bidirectionally transports three Na⁺ ions for one Ca²⁺ ion across the sarcolemmal membrane,¹ the direction of the exchange being dependent on the membrane potential and the intracellular and extracellular Na⁺ and Ca²⁺ concentrations.² Mainly, the Na⁺-Ca²⁺ exchanger has been regarded as a Ca²⁺ extrusion system contributing to the maintenance of low diastolic Ca²⁺ levels and cardiac relaxation.^{3 4 5} Apparently, it is responsible for the extrusion of 20% of the Ca²⁺ from the cytosol during diastole, whereas the second Ca²⁺ extrusion system, the sarcoplasmic reticulum Ca²⁺-ATPase, serves for 80% of Ca²⁺ sequestration from the cytosol into the sarcoplasmic reticulum.⁶ However, there is experimental evidence that the Na⁺-Ca²⁺ exchanger also serves as a source for Ca²⁺ influx into the cell.^{2 3 7 8} This "reverse mode" Na⁺-Ca²⁺ exchange can be enhanced by decreasing the transsarcolemmal Na⁺ gradient or by increasing the transsarcolemmal Ca²⁺ gradient.² Several studies have provided evidence that reverse mode Na⁺-Ca²⁺ exchange may possess a possible role in inducing sarcoplasmic reticulum Ca²⁺ release and modulation of cardiac contractility.^{9 10 11 12 13 14 15}

In cardiac hypertrophy and failure, essential alterations in sarcoplasmic reticulum function have been described.^{16 17} An impaired diastolic uptake of Ca²⁺ into the sarcoplasmic reticulum^{18 19 20} and a decreased activity of the sarcoplasmic reticulum Ca²⁺-ATPase²¹ could be responsible for major changes in cardiac Ca²⁺ handling, especially an increase in diastolic intracellular Ca²⁺ levels,¹⁷ a decrease of the peak Ca²⁺-transient during systole,¹⁷ a prolonged diastolic decline of the intracellular Ca²⁺ concentration,¹⁷ and an impaired diastolic relaxation.²² Interestingly, for the alternative Ca²⁺ extrusion system, the sarcolemmal Na⁺-Ca²⁺ exchanger, increased activity has been observed in some animal models of arterial hypertension²³ and cardiac hypertrophy.^{24 25 26} Only recently, Studer et al²⁷ demonstrated that left ventricular mRNA and protein levels of the Na⁺-Ca²⁺ exchanger are increased in human idiopathic dilated cardiomyopathy. The functional relevance of this finding has remained obscure thus far.

The present study aimed at providing the missing evidence for the functional relevance of an enhanced expression of the Na⁺-Ca²⁺ exchanger. Therefore, molecular and biochemical experiments were combined with functional data obtained in the same laboratory, and an overview was provided of recent data in the literature explaining the mechanism by which Na⁺-Ca²⁺ exchange modulates cardiac contractility. Steady-state mRNA and protein levels of the Na⁺-Ca²⁺ exchanger were determined in failing human left ventricular myocardium from patients with end-stage heart failure due to dilated cardiomyopathy or ischemic heart disease and in nonfailing human left ventricular myocardium from organ donors. In addition, the effects of the Na⁺ channel activator BDF 9148 on cardiac force of contraction, on diastolic tension, and on the force-frequency relationship in failing and nonfailing left ventricular myocardium were examined. BDF 9148 has been known to increase the intracellular Na⁺ concentration by augmenting the open state of sarcolemmal Na⁺ channels.²⁸ The resulting positive inotropic response has been suggested to be mediated by an enhancement of the activity of the Na⁺-Ca²⁺ exchanger.^{29 30} In contrast to the decreased contractile response of the failing myocardium to cAMP-dependent inotropic agents,^{31 32 33 34} the inotropic efficacy of Na⁺ channel activators is surprisingly unchanged in failing myocardium.^{35 36} The hypothesis for this study was that if an increased expression of the Na⁺-Ca²⁺ exchanger in the failing myocardium is of functional relevance, one would expect that this might have consequences for the inotropic response of the failing and the nonfailing myocardium to BDF 9148. Since the exchanger serves for both Ca²⁺ extrusion and Ca²⁺ influx and both mechanisms are influenced by changes in the intracellular Na⁺ concentration, special emphasis was laid on the question of whether BDF 9148 is able to improve mainly the systolic or the diastolic inotropic performance or is able to improve both.

	Methods

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Human Myocardial Tissue
Experiments were performed on failing myocardium from 8 patients with idiopathic dilated cardiomyopathy, from 6 patients with ischemic cardiomyopathy, and on nonfailing myocardium from 8 organ donors whose hearts could not be used for transplantation. Myocardial tissue was obtained during cardiac transplantation. All patients gave written informed consent before cardiac surgery. Medical therapy of the patients with idiopathic dilated cardiomyopathy consisted of cardiac glycosides, diuretics, and nitrates. All patients with cardiomyopathy also received ACE inhibitors. Patients who received catecholamines or ß-adrenoceptor antagonists were withdrawn from the study. The hemodynamic and biographical data are given in Table 1. Patient history of the organ donors (4 men, 4 women; age, 50.9±1.99 years) and two-dimensional echocardiography revealed no evidence of heart disease. General anesthesia was performed with flunitrazepam, fentanyl, and pancuronium bromide with isoflurane. Cardiac surgery was performed with the patient on cardiopulmonary bypass. Tissue procurement of diseased myocardium and nonfailing myocardium was the same. Only noninfarcted tissue was used, and scars were carefully trimmed away. Tissue pieces were suspended in ice-cold cardioplegic solution (modified Bretschneider solution containing [in mmol/L] NaCl 15, KCl 10, MgCl₂ 4, histidine HCl 180, tryptophane 2, mannitol 30, and potassium dihydrogen oxoglutarate 1) and were delivered from the operating room to the laboratory within 5 minutes.

View this table: [in this window] [in a new window]   Table 1. Biographical and Hemodynamic Data of Patients With Heart Failure

Isolation of Total RNA
Total RNA from frozen left ventricular tissue samples was prepared according to the protocol of Chomczynski and Sacchi.³⁷ Typically, between 50 and 100 µg of total RNA was obtained from 150 mg tissue. The amount of RNA was determined by UV absorption. The optical density ratio of 260 to 280 nm was 1.8 to 2.0 in all cases.

Northern Blot Analysis
Total RNA (10 µg) was separated in a 6% formaldehyde/1.2% agarose gel, blotted onto nylon membranes (Schleicher and Schuell) by overnight capillary blotting, and fixed by UV irradiation. Complete mRNA transfer to the nylon membrane had been controlled by ethidium bromide staining of the gel. After fixation, the blots were prehybridized in 50% formamide solution (5x SSC, 5x Denhardt's solution, 50% formamide, 1% SDS, 50 mmol/L sodium phosphate, pH 6.8, 10% dextran sulfate, 1 mg/mL salmon sperm DNA). Hybridization was performed in 50% formamide solution at 42°C for at least 16 hours. The membrane was successively hybridized with a 500-bp cDNA fragment (EcoRI-EcoRI) encoding rat heart Na⁺-Ca²⁺ exchanger³⁸ (kindly donated by Prof K.D. Philipson, PhD, Los Angeles, Calif), a 680-bp cDNA fragment (PstI-PstI) encoding human atrial natriuretic factor (ANF)³⁹ (kindly donated by Suntory Limited, Osaka, Japan), and a 260-bp cDNA fragment encoding human ß-myosin heavy chain (ß-MHC) (kindly provided by Paul Barton, PhD, London, United Kingdom). The fragments had been cut out from the plasmid vector with the appropriate restriction enzymes, separated from the vector DNA on a 1% low-melt agarose gel, and labeled with [-³²P]dCTP (Amersham Buchler Ltd) with the use of the Multiprime DNA labeling kit from Amersham Buchler Ltd. The activity of the respective labeled probe in the hybridization solution was 1x10⁶ cpm/mL. After hybridization at 42°C overnight, the membrane was successively washed twice in 2x SSC/0.1% SDS at room temperature for 15 minutes and twice in 0.2x SSC/0.1% SDS at 68°C for 45 minutes. Standardization was performed by hybridization of the same membrane with the use of a 40-bp, single-stranded synthetic oligonucleotide probe for GAPDH (Dianova). Hybridization conditions were the same as described above. Stringency washes were performed with 2x SSC/0.1% SDS at room temperature, for 30 minutes with 2x SSC/0.1% SDS at 65°C, and twice for 5 minutes with 2x SSC/0.1% SDS at room temperature. Membranes were exposed to Kodak films (X-OMAT). Quantification of the signals was performed by densitometric analysis with the use of the Image Quant Densitometric System (Molecular Dynamics). Before the final experiments, we ensured that the amount of RNA loaded on the gel as well as the cDNA concentration in the labeling buffer and the exposition time of the photographic film were optimized for obtaining hybridization signals, which were within the linear range.

Myocardial Protein Preparation
Myocardial tissue (3 to 5 g) was thawed on ice and chilled in 10 mL of ice-cold homogenization buffer (Tris-HCl 20 mmol/L, EDTA 1 mmol/L, and dithiothreitol 1 mmol/L, pH 7.4). Connective tissue was trimmed away, and tissues were minced with scissors; the membranes were homogenized by hand for 1 minute with a glass-glass homogenizer. The suspension was centrifuged at 100 000g for 30 minutes. The supernatant was discarded, and the pellet was resuspended in 10 mL of homogenization buffer and recentrifuged at 100 000g for 30 minutes. Finally, the remaining pellet was dissolved in 5 mL of the same buffer.

Immunoblotting Technique
Immunoblotting techniques were performed as previously described with slight modifications.³⁴ Myocardial proteins were denatured by heating to 95°C in electrophoresis buffer containing 4% SDS, 50 mmol/L Tris-HCl, pH 6.8, 20% glycerol, 0.05% pyronin, and 10 mmol/L dithiothreitol. Separation of proteins was performed by SDS-PAGE (7% [wt/vol] acrylamide, 16-cm length). The amount of protein used in the definite experiments was 50 µg, which allowed quantitative Na⁺-Ca²⁺ exchanger protein analysis within the linear range. After electrophoretic separation, proteins were transferred to a nitrocellulose membrane (0.2 µm) by semidry electrophoretic blotting with the use of an LKB 2117-250 Novablot electrophoretic transfer kit (LKB-Pharmacia GmbH) (0.8 mA/cm², 90 minutes) with a discontinuous buffer system according to the method of Towbin et al.⁴⁰ Complete electrophoretic transfer was controlled by staining the nitrocellulose membranes with Ponceau red and staining the gel with Coomassie blue, and it was ensured that there was no loss of protein caused by overblotting. The sheets were immersed in 100 mL of 5% low-fat dry milk powder in PBS buffer (KH₂PO₄ 100 mmol/L, NaCl 137 mmol/L, KCl 2.68 mmol/L, NaH₂PO₄xH₂O 10.44 mmol/L, pH 7.4) and shaken for at least 1 hour at room temperature. After repeated washes in PBS/Tween-20, the sheets were incubated with the first antibody. Na⁺-Ca²⁺ exchanger protein levels were determined in myocardial samples with the use of a rabbit antiserum raised against the canine Na⁺-Ca²⁺ exchanger (diluted 1:500),⁴⁰ which was purchased from Swant. For detection of ß-MHC, a monoclonal specific antibody raised in mouse (diluted 1:500)⁴² was purchased from Biocytex. After repeated washes in PBS, immunoreaction was continued by incubation of the nitrocellulose sheets with a peroxidase-conjugated goat secondary antibody (diluted 1:70 000) raised against rabbit and mouse IgG, respectively. After further washes, detection was performed with the enhanced chemiluminescence assay (ECL kit). After exposure to x-ray film (Kodak X-OMAT AR), signals were quantified by two-dimensional densitometry (Image Quant Densitometric System, Molecular Dynamics).

Immunohistochemical Staining
For immunohistochemical localization of Na⁺-Ca²⁺ exchanger protein within the left ventricular myocardium, deparaffinized tissue sections were incubated for 30 minutes in Tris-buffered saline (TBS, pH 7.4). The primary antiserum, rabbit anti-dog cardiac sarcolemmal Na⁺-Ca²⁺ exchanger,⁴⁰ was diluted 1:1000 in TBS containing 2.5% BSA and 0.1% sodium azide (SA-TBS, pH 7.6) and placed over the tissue sections in a humid chamber at 4°C overnight. The sections were brought to room temperature for successive incubations in TBS (10 minutes), biotinylated swine anti-rabbit -globulin (1:5000 in normal human serum, 30 minutes), TBS (10 minutes), streptavidin conjugated to alkaline phosphatase (1:50 in SA-TBS), TBS (2 times, 10 minutes), and 0.1% fast red TR salt in TBS (pH 8.2) containing 0.023% levamisole and 1:50 of a 1% solution of naphthol-AS-MX phosphate in dimethylformamide (up to 30 minutes, according to microscopic control). The sections were rinsed with TBS and then were counterstained with hematoxylin-eosin.

Isolated Cardiac Muscle Strip Preparation and Measurement of Force of Contraction
Immediately after excision, the papillary muscles were placed in ice-cold, preaerated Tyrode's solution. The experiments were performed on isolated, electrically driven muscle preparations. Muscle strips of uniform size with muscle fibers running approximately parallel to the length of the strips were dissected under microscopic control with scissors in aerated, modified Tyrode's solution (composition below). Connective tissue was carefully trimmed away. The muscles were suspended in an organ bath (75 mL) maintained at 37°C and containing a modified Tyrode's solution of the following composition (in mmol/L): NaCl 119.8, KCl 5.4, MgCl₂ 1.05, CaCl₂ 1.8, NaHCO₃ 22.6, NaH₂PO₄ 0.42, glucose 5.0, ascorbic acid 0.28, and EDTA 0.05. The bathing solution was continuously aerated with 95% O₂ and 5% CO₂. The muscles were stimulated by two platinum electrodes with the use of field stimulation from a Grass S88 stimulator (frequency, 1 Hz; impulse duration, 5 ms; intensity, 10% to 20% greater than threshold). Each muscle was stretched to the length at which force of contraction was maximal. The developed tension was measured isometrically with an inductive force transducer (W. Fleck) attached to a Gould recorder. Preparations were allowed to equilibrate for at least 90 minutes, with the bathing solution changed once after 45 minutes. Experimental details have been described elsewhere.⁴³

Miscellaneous
Protein was determined according to the method of Lowry et al⁴⁴ with the use of BSA as standard. SDS-PAGE was performed as described by Lammli.⁴⁵ 5'-Nucleotidase activity was analyzed with the method of Dixon and Purdom.⁴⁶

Materials
BDF 9148 (4-[3-(1-Diphenylmethyl-azetidin-3-oxy)-2-hydroxy-propoxy]-1H-indol-2-carbonitril) was kindly provided by Beiersdorf AG. Isoprenaline and antibodies, if not indicated otherwise, were from Sigma-Aldrich. Restriction enzymes were purchased from Boehringer. All other chemicals were of analytical grade or the best grade commercially available. Only deionized and double-distilled water was used throughout.

Statistics
Data shown are mean±SEM. EC₅₀ values were obtained by graphic evaluation of the pD₂ values of the half-maximal effect of each individual concentration-response curve and are shown as means with the 95% confidence limit. Statistical significance was estimated with the Student's t test for unpaired observations and ANOVA according to Wallenstein et al.⁴⁷ A value of P<.05 was considered significant. K_d values were determined graphically in each individual experiment.

	Results

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mRNA Steady-state Levels of Na⁺-Ca²⁺ Exchanger
Quantification of Na⁺-Ca²⁺ exchanger mRNA steady-state levels in left ventricular myocardium from failing and nonfailing hearts was performed by Northern blot analysis with a 500-bp cDNA fragment (EcoRI-EcoRI) encoding the rat heart Na⁺-Ca²⁺ exchanger. The nucleotide sequence of the cDNA fragment showed a 90% homology to the human Na⁺-Ca²⁺ exchanger cDNA sequence published by Izumo et al.⁴⁸ After 5 days of exposure, a single hybridization band was obtained at the expected position, corresponding to a mRNA size of 7.2 kb. Representative hybridization signals are shown in Fig 1. The strongest signal was obtained in failing myocardium with idiopathic dilated cardiomyopathy. A similarly enhanced signal was obtained from ischemic cardiomyopathy hearts. Only a weak signal was observed in nonfailing myocardium. Values obtained from densitometric analysis were related to densitometric values of the hybridization signals of GAPDH, which was used as a control for the amount of RNA loaded on the gel. Mean values of relative levels of Na⁺-Ca²⁺ exchanger mRNA are given in Table 2. There was a significant increase in Na⁺-Ca²⁺ exchanger mRNA levels, by 79% (P<.001) in dilated cardiomyopathy and by 58% (P<.05) in ischemic cardiomyopathy compared with nonfailing hearts (Fig 1). The result was not significantly altered when Na⁺-Ca²⁺ exchanger mRNA levels were related to ß-MHC mRNA levels as a marker for the amount of contractile proteins (idiopathic dilated cardiomyopathy increased by 58%, P<.01, ischemic cardiomyopathy increased by 27%, P<.05). mRNA levels for ß-MHC were not significantly different in the failing and the nonfailing myocardium (Table 2).

View larger version (44K): [in this window] [in a new window]   Figure 1. Northern blot analysis of Na+-Ca2+ exchanger mRNA. Representative hybridization signals obtained from failing human left ventricular myocardium due to dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) and from nonfailing myocardium (NF) are shown. Bar graph shows mean values related to GAPDH (±SEM) for the three groups, obtained by densitometric analysis.

View this table: [in this window] [in a new window]   Table 2. mRNA Steady-state Levels of Na+-Ca2+ Exchanger, ß-Myosin Heavy Chain, and Atrial Natriuretic Factor in Relation to GAPDH in Nonfailing and Failing Human Left Ventricular Myocardia as Obtained by Northern Blot Analysis

Protein Levels of Na⁺-Ca²⁺ Exchanger
Immunochemical detection of Na⁺-Ca²⁺ exchanger protein levels by Western blot analysis with the use of a specific polyclonal antibody directed against the canine Na⁺-Ca²⁺ exchanger revealed two bands, one at the expected position corresponding to 120 kD and the other at a position corresponding to 70 kD. The immunoreactive protein pattern found in human myocardium is identical to that obtained by Western blot analysis of the canine myocardium,⁴¹ indicating that the antibody specifically reacts with the human cardiac Na⁺-Ca²⁺ exchanger in the same way as with the canine cardiac exchanger. According to previous reports, the second band is regarded as a proteolytic product of the entire protein⁴¹ ; therefore, for quantification of Na⁺-Ca²⁺ exchanger protein levels, densitometric values of both bands were added. As can be seen in Fig 2, the two immunoreactive proteins observed in nonfailing myocardium were much weaker than those detected in failing myocardium. Mean values of the densitometric analysis of the Na⁺-Ca²⁺ exchanger protein bands are given in Table 3. In dilated cardiomyopathy and ischemic cardiomyopathy, mean values were increased by 35.5% (P<.001) and 20.4% (P<.05), respectively, compared with nonfailing myocardium. The results were not different when the immunoreactive bands were determined separately or were added (not shown). The result was not significantly altered when Na⁺-Ca²⁺ exchanger protein levels were related to ß-MHC protein levels, which were also determined by Western blot analysis and were used as a marker for the amount of contractile proteins, or when Na⁺-Ca²⁺ exchanger protein levels were related to 5'-nucleotidase activity, which serves as a marker for the amount of membrane protein (Table 3).

View larger version (30K): [in this window] [in a new window]   Figure 2. Immunochemical detection (Western blots) of Na+-Ca2+ exchanger protein in failing and in nonfailing human left ventricular myocardium performed by 7% SDS-PAGE. Representative signals obtained from failing human left ventricular myocardium due to dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) and from nonfailing myocardium (NF) are shown. Bar graph shows mean values (±SEM) for the three groups, obtained by densitometric analysis.

View this table: [in this window] [in a new window]   Table 3. Protein Concentrations of the Sarcolemmal Na+-Ca2+ Exchanger in Nonfailing and Failing Human Left Ventricular Myocardium

Immunohistochemical Staining
Both the cDNA probe and the antibody used in this study are not specific for the cardiac isoform of the Na⁺-Ca²⁺ exchanger. To ensure that the observed alterations on myocardial Na⁺-Ca²⁺ exchanger protein levels reflect changes in the cardiac myocytes, we performed immunohistochemical staining using the above-described polyclonal antibody raised against the canine exchanger protein. As shown in Fig 3, immunolocalization exhibited a positive diffuse staining of cardiac myocytes. At high-power views, the reaction product specifically outlined the sarcolemmal membrane (Fig 3b) and the T-tubule system (Fig 3c), which is in accordance with recent findings.^{49 50} There was a cross-reaction of the antibody with vascular smooth muscle cells of vessel walls, but in general smooth muscle cells showed only a faint positive staining. Other cells such as fibrocytes or endothelial cells were not stained by the Na⁺-Ca²⁺ exchanger antibody. We ensured specificity of the reaction by staining left ventricular tissue slices using the same procedure but leaving out the first specific antibody (Fig 3d and 3e).

View larger version (116K): [in this window] [in a new window]   Figure 3. Immunohistochemical staining of left ventricular myocardial slices with the use of a polyclonal antiserum raised in rabbit against the canine Na+-Ca2+ exchanger. Note that mainly cardiac myocytes are stained in comparison with less intense staining of vascular smooth muscle cells and absent staining of the connective tissue and capillary endothelia (magnification x480, a). In the high-power view (magnification x1700), a delicate labeling of the sarcolemmal membrane (arrow) (b) and the T-tubule system at their invagination points (arrows) (c) is discernible. Control staining of an adjacent tissue section according to the same procedure omitting the specific immune serum is shown in d (magnification x480). Lacking specific immunostaining, the sarcolemmal membrane (arrows) is barely perceptible (e and f, magnification x1700).

mRNA Levels of Atrial Natriuretic Factor
We performed Northern blot analysis of ANF to ensure that the failing myocardium examined in this study revealed changes of gene expression typical for heart failure. Northern blot analysis of ANF revealed a single hybridization signal at a position corresponding to a mRNA size of 900 bp. In general, the ANF mRNA signal observed in failing myocardium due to idiopathic dilated cardiomyopathy was stronger than the signal obtained from failing myocardium due to ischemic heart disease. There was hardly any signal in the nonfailing myocardium. Representative signals and mean values of failing and nonfailing myocardium are shown in Fig 4 and Table 2.

View larger version (25K): [in this window] [in a new window]   Figure 4. Northern blot analysis of atrial natriuretic factor (ANF) mRNA. Representative hybridization signals obtained from failing human left ventricular myocardium due to dilated cardiomyopathy (DCM) and ischemic cardiomyopathy (ICM) and from nonfailing myocardium (NF) are shown. The hybridization signal for GAPDH is shown as a control. Bar graph shows mean values (±SEM) for the three groups, obtained by densitometric analysis.

Effects of Isoprenaline and BDF 9148
Fig 5 shows the positive inotropic effects of isoprenaline on isolated electrically driven papillary muscle strips from nonfailing and failing human myocardium. Isoprenaline increased the force of contraction in a concentration-dependent manner in both groups. The positive inotropic effect was significantly decreased in failing compared with nonfailing myocardium, as was the potency of isoprenaline, indicated by EC₅₀ values of 0.016 (0.01 to 0.02) µmol/L and 0.06 (0.04 to 0.08) µmol/L, respectively (P<.05).

View larger version (20K): [in this window] [in a new window]   Figure 5. Concentration-response curves for the effects of isoprenaline (0.0001 to 1 µmol/L) on isometric force of contraction of isolated, electrically driven papillary muscle strips from nonfailing donor hearts (NF, n=8) and from failing hearts from patients who had undergone cardiac transplantation (NYHA class IV, n=8). Ordinates: Increase in force of contraction in mN, increase in force of contraction in % of maximal effect; abscissae: concentration of isoprenaline in µmol/L.

The positive inotropic effect of the Na⁺ channel activator BDF 9148 is demonstrated in Fig 6. BDF 9148 (0.03 to 30 µmol/L) increased the force of contraction in failing and nonfailing papillary muscle strips in a concentration-dependent manner. In contrast to isoprenaline, the maximal positive inotropic effect of BDF 9148 was identical in failing (6.5±0.4 mN) and in nonfailing (6.2±0.3 mN) myocardium. The potency of BDF 9148 to increase the force of contraction was significantly higher in failing than in nonfailing myocardium, as judged by the EC₅₀ values, which were significantly lower (P<.01) in failing (0.175 [0.155 to 0.22] µmol/L) compared with nonfailing myocardium (1.65 [1.3 to 3.0] µmol/L) (Table 4).

View larger version (18K): [in this window] [in a new window]   Figure 6. Concentration-response curves for the effects of BDF 9148 (0.03 to 30 µmol/L) on isometric force of contraction of isolated, electrically driven papillary muscle strips from nonfailing donor hearts (NF, n=8) and from failing hearts from patients who had undergone cardiac transplantation (NYHA class IV, n=8). Ordinates: Increase in force of contraction in mN, increase in force of contraction in % of maximal effect; abscissae: concentration of BDF 9148 in µmol/L.

View this table: [in this window] [in a new window]   Table 4. Effect of Increasing Concentrations of BDF 9148 on Developed Force of Contraction in Isolated Human Papillary Muscle Strips

Within the concentration range from 0.1 to 30 µmol/L, BDF 9148 increased diastolic tension neither in the nonfailing (not shown) nor in the failing myocardium (Fig 7). No significant difference between groups could be observed.

View larger version (24K): [in this window] [in a new window]   Figure 7. Concentration-response curves for the effect of BDF 9148 (0.03 to 30 µmol/L) on diastolic tension and on maximally developed tension of isolated, electrically driven papillary muscle strips from failing hearts (NYHA class IV, n=8). Ordinate: Change in tension in mN; abscissa: concentration of BDF 9148 in µmol/L.

Fig 8 shows the frequency-dependent increase in force of contraction in nonfailing and failing human myocardium in the absence and the presence of BDF 9148 (frequency range from 0.5 to 3 Hz). As expected, the nonfailing myocardium responded to the increase in the stimulation frequency with a significant increase in force of contraction. BDF 9148 (0.1 µmol/L) did not significantly alter the frequency-response curve in the nonfailing myocardium. In contrast, in the failing myocardium in the absence of BDF 9148, the increase in stimulation frequency was accompanied by a reduction in the force of contraction. Addition of BDF 9148 in a concentration that had no significant inotropic effect (0.1 µmol/L) reversed the negative force-frequency relationship in the failing myocardium. Increasing the stimulation rate then led to an increase in force of contraction that was similar to the frequency-dependent increase in the nonfailing myocardium (Table 5). In the failing myocardium, the frequency-dependent increase in diastolic tension was not increased in the presence of BDF 9148 (1 µmol/L) but rather slightly decreased (Fig 9). Even more, the ratio between the frequency-dependent increase in diastolic tension and the developed force of contraction was significantly reduced in the presence of BDF 9148 (Fig 9).

View larger version (24K): [in this window] [in a new window]   Figure 8. Force-frequency relationship under basal conditions and after stimulation with BDF 9148 (0.1 µmol/L) on isolated, electrically driven papillary muscle strips from nonfailing (n=5) and terminally failing hearts (NYHA class IV, n=5). Ordinates: Force of contraction in mN; abscissae: frequency of stimulation in Hz.

View this table: [in this window] [in a new window]   Table 5. Effect of Increasing Stimulation Frequencies in Absence and in Presence of BDF 9148 (1 µmol/L) on Developed Force of Contraction in Isolated Human Papillary Muscle Strips

View larger version (25K): [in this window] [in a new window]   Figure 9. Frequency-dependent increase in diastolic tension and frequency-dependent increase in diastolic tension related to force of contraction under basal conditions and after stimulation with BDF 9148 (0.1 µmol/L) in isolated, electrically driven papillary muscle strips from terminally failing hearts (n=5, bottom). Ordinates: Top, change in diastolic tension in mN; bottom, change in diastolic tension related to force of contraction in mN; abscissae: frequency of stimulation in Hz.

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
In failing compared with nonfailing human left ventricular myocardium, a significant increase in Na⁺-Ca²⁺ exchanger mRNA and protein levels was observed. This increase was significant regardless of whether Na⁺-Ca²⁺ exchanger mRNA levels were related to GAPDH or to ß-MHC mRNA levels and whether protein levels were related to total protein content, to ß-MHC protein content as a marker for the amount of contractile proteins, or to 5'-nucleotidase activity as a marker for the amount of membrane proteins. Thus, the observed increase in Na⁺-Ca²⁺ exchanger mRNA and protein levels is not simply due to changes in the amount of other myocardial proteins or to an altered myocyte-nonmyocyte ratio. It indicates a specific increase in the amount of exchanger molecules. Immunohistochemical staining of myocardial tissue showed that there was no cross-reaction of the antibody used in this study with noncontractile cells such as endothelial cells and fibrocytes. There was a cross-reaction of the Na⁺-Ca²⁺ exchanger antibody with vascular smooth muscle cells. However, the amount of smooth muscle cells is rather negligible compared with the amount of cardiac myocytes. Therefore, one should assume that the increase in Na⁺-Ca²⁺ exchanger protein levels is due to alterations within cardiac myocytes. The enhanced expression of ANF in the failing left ventricular myocardium⁵¹ and the decreased concentration-dependent inotropic response of the failing myocardium to isoprenaline^{31 32 33 34} together with the clinical data of the patients demonstrate that the observations were made in myocardium that revealed typical molecular, biochemical, and functional characteristics of failing and nonfailing hearts. Since the increase in Na⁺-Ca²⁺ exchanger mRNA and protein levels is similar in idiopathic dilated cardiomyopathy and in ischemic heart disease, it appears to be independent from the underlying pathophysiology of the disease.

Recently, Studer et al²⁷ reported that in failing human left ventricular myocardium, Na⁺-Ca²⁺ exchanger mRNA and protein levels are increased, whereas Ca²⁺ATPase mRNA and protein levels in sarcoplasmic reticulum are decreased. Their observation favored the suggestion that the increase in Na⁺-Ca²⁺ exchanger mRNA and protein levels in the failing myocardium could be a compensatory mechanism for a decreased uptake of Ca²⁺ into the sarcoplasmic reticulum^{18 19 20} and a reduced activity of Ca²⁺ATPase in sarcoplasmic reticulum.²¹ However, since the Na⁺-Ca²⁺ exchanger also serves as a gate for Ca²⁺ entry into the myocyte,² the enhanced expression of the exchanger also could be of functional relevance because it leads to an increased influx of Ca²⁺.

The functional experiments performed in this study for the first time demonstrate that the increased expression of the Na⁺-Ca²⁺ exchanger is of importance for the contractile performance of the failing heart. In both nonfailing and failing myocardium, the Na⁺ channel agonist BDF 9148 increased the force of contraction in a concentration-dependent manner. Interestingly, the potency of BDF 9148 was significantly higher in the failing than in the nonfailing myocardium. Only recently, it has been demonstrated that the inotropic effect of BDF 9148 in human myocardium is accompanied by a prolongation of the action potential duration and a prolongation of the course of contraction.⁵² Thus, the increase in action potential duration itself could explain the increase in force of contraction. However, it is generally accepted that Na⁺ channel agonists exert their positive inotropic effect by increasing the intracellular Na⁺ concentration, which consequently leads to an increase in the activity of the Na⁺-Ca²⁺ exchanger.^{28 29 30 52} Therefore, it is most likely that the increased expression of the exchanger in the failing myocardium is responsible for the increased inotropic potency of BDF 9148.

There are different mechanisms by which enhancement of the exchanger activity in response to a Na⁺ channel activator could lead to an increase in force of contraction. An increase in the intracellular Na⁺ concentration reduces the driving force for the extrusion of Ca²⁺ from the cell and possibly increases the influx of Ca²⁺ via the exchanger.^{2 7} Both would result in a net decrease of Ca²⁺ extrusion from the myocyte, possibly in an increased loading of Ca²⁺ into the sarcoplasmic reticulum and thereby in an increase in cardiac force of contraction.⁵³ Alternatively, increased Ca²⁺ influx via the exchanger could modulate cardiac force of contraction directly¹⁰ or by triggering sarcoplasmic reticulum Ca²⁺ release.¹⁴ Only recently, Lipp and Niggli⁵⁴ demonstrated that Ca²⁺ release from the sarcoplasmic reticulum could be triggered either by the Ca²⁺ current or the Na⁺ current.

An increased influx of Ca²⁺ via the exchanger because of an increase in exchanger molecules would be the most likely explanation for BDF 9148 being more potent in the failing than in the nonfailing myocardium. An alternative hypothesis would be a difference in the number of sarcolemmal Na⁺ channels in failing and nonfailing myocardium. Thus far, there is no experimental evidence for such an alteration. Data concerning the amount of sarcolemmal Ca²⁺ channels in failing and hypertrophied myocardium are contradictory insofar as an increase,⁵⁵ a decrease,⁵⁶ and no changes⁵⁷ compared with nonfailing myocardium have been described. However, the Ca²⁺ current density has been reported to be unchanged in failing myocardium.¹⁷ Therefore, the density of sarcolemmal Ca²⁺ channels is a rather unlikely explanation for the increased potency of BDF 9148 in the failing myocardium.

BDF 9148, at a low concentration that had no positive inotropic effect, restored the inversed force-frequency relationship in the failing myocardium described before.^{58 59} The force-frequency relationship of the nonfailing myocardium was not altered by BDF 9148. These findings are in accordance with previous data⁶⁰ demonstrating a similar effect of ouabain in combination with BDF 9148. The frequency-dependent increase in cardiac force of contraction is possibly mediated by an increase in the intracellular Na⁺ concentration,⁶¹ which results in a net influx of Ca²⁺ into the myocyte.⁶² In accordance with this theory, Harrison and Boyett⁶³ recently demonstrated that increasing the stimulation frequency leads to a net increase in outward Na⁺-Ca²⁺ exchange current (net Ca²⁺ influx) in response to a rise in the intracellular Na⁺ concentration in guinea pig ventricular myocytes. This Ca²⁺ influx augments the Ca²⁺ load of the cell and could thereby contribute to the rate-dependent increase in force of contraction. The altered force-frequency relationship in the failing myocardium is likely to be caused by an impairment of the intracellular Ca²⁺ handling,⁶⁴ especially by a decreased uptake of Ca²⁺ into the sarcoplasmic reticulum.^{19 65} However, one could also speculate that an altered availability of intracellular Na⁺ is responsible for the "negative treppe" phenomenon in the failing myocardium. The resulting inadequate activation of the Na⁺-Ca²⁺ exchanger and an insufficient net Ca²⁺ influx would explain the "negative treppe" phenomenon. Accordingly, the positive effect of BDF 9148 on the force-frequency relationship in the failing myocardium could result from an increased Ca²⁺ influx via the exchanger in response to an improved Na⁺ availability. The observation that the effect of BDF 9148 is frequency dependent is in accordance with previous reports about the frequency dependence of other Na⁺ channel activators such as veratridine⁶⁶ or DPI 201-106, from which BDF 9148 is derived.⁶⁷

One might expect that an increase in the intracellular Na⁺ concentration favoring Ca²⁺ influx via the Na⁺-Ca²⁺ exchanger might limit Ca²⁺ extrusion via this system. This Ca²⁺ gain would be expected to increase diastolic tension. Especially if the enhanced expression of the Na⁺-Ca²⁺ exchanger is an essential compensatory mechanism for Ca²⁺ sequestration in the failing myocardium, the reduction of Ca²⁺ extrusion via the exchanger should have adverse effects on cardiac contractility, in particular on diastolic function. Interestingly, this does not appear to be the case. Increasing concentrations of BDF 9148 did not lead to an increase in diastolic tension either in failing or nonfailing myocardium. The positive effect of BDF 9148 on the frequency-dependent increase in force of contraction in the failing myocardium was not accompanied by a frequency-dependent increase in diastolic tension. In contrast, in the presence of BDF 9148, the ratio between the frequency-dependent increase in diastolic tension and the increase in developed force of contraction was significantly lowered in the failing myocardium. Thus, BDF 9148 appears to combine positive inotropic and lusitropic effects. The benefit of a gain in force of contraction caused by Na⁺ channel modulation is not eliminated by a negative effect on diastolic relaxation. Future studies providing electrophysiological data on the effect of BDF 9148 will be necessary to explain this phenomenon.

The fact that diastolic tension is not influenced negatively by BDF 9148 could suggest that the extent to which the diastolic cytosolic Ca²⁺ concentration is altered is too small to be of relevance for diastolic tension. This hypothesis is in agreement with the theory of Lederer et al,⁶⁸ who postulated a "fuzzy space," which is a region in which Na⁺ channels, the Na⁺-Ca²⁺ exchanger, sarcolemmal Ca²⁺ channels, and sarcoplasmic reticulum Ca²⁺ release channels are in close proximity. According to this theory, local slow diffusion of Na⁺ and Ca²⁺ ions that does not affect mean intracellular ion concentrations could be sufficient for triggering sarcoplasmic reticulum Ca²⁺ release. Also, the increase in the cytosolic Ca²⁺ concentration could be compensated by the sarcoplasmic reticulum, which would also explain the increase in force of contraction. An alternative theory has been presented by Bers,⁹ who examined the effect of acetylstrophantidin on force of contraction and on net transsarcolemmal Ca²⁺ fluxes in rabbit cardiac muscles. According to his observations, the reduction of the Na⁺ gradient could lead to an increase of both Ca²⁺ influx and, especially at potentiated contractions, of Ca²⁺ efflux. This increase in Ca²⁺ efflux could be secondary to an enhanced release of Ca²⁺ from the sarcoplasmic reticulum.⁹ Following this theory, the restoration of the force-frequency relationship in the failing heart by BDF 9148 may be due to both enhanced Ca²⁺ influx and perhaps increased sarcoplasmic reticulum Ca²⁺ load and also enhanced Ca²⁺ extrusion via the exchanger leading to an increase of the Ca²⁺ gradient.

Considering the potential therapeutic use of Na⁺ channel activators in chronic heart failure, a major hazard may be that Na⁺ channel modulation is connected with potential proarrhythmogenic effects. It is known that alterations to Na⁺ channel function could delay cardiac repolarization, which could lead to QT prolongation and subsequent malignant arrhythmias such as Torsade de pointes.^{69 70 71} Prolongation of the QT interval has been observed for DPI 201-106 in humans.⁶⁷ Only recently it has been shown that the inherited long QT syndrome causing sudden death from cardiac arrhythmias is associated with mutations of the Na⁺ channel gene SCN5a within a region, which is important for Na⁺ channel inactivation.⁷² In this context, it may be encouraging that in dogs, the frequency-corrected QT interval was less prolonged with BDF 9148 than with DPI 201-106.⁷³ Baumgart et al⁷⁴ demonstrated that in anesthetized open chest dogs, the application of BDF 9148 in a dosage that exerted a profound positive inotropic effect was not accompanied by severe proarrhythmogenic side effects. However, effects in humans may be different. It has already been shown that in contrast to different effects in guinea pig papillary muscle,⁷⁵ BDF 9148 leads to a prolongation of the action potential in human papillary muscle, which is similar to the effect of DPI 201-106.⁵² Because of this finding, one might speculate whether it also could lead to a prolongation of the QT interval and to subsequent malignant arrhythmias.

Conclusions
The data of this study demonstrate that the increased expression of the sarcolemmal Na⁺-Ca²⁺ exchanger is accompanied by a more pronounced contractile response to the Na⁺ channel agonist BDF 9148. Enhancement of the Na⁺-Ca²⁺ exchanger activity by the Na⁺ channel agonist leads to an increase in systolic force of contraction, whereas diastolic tension is unaffected or even improved. This may be of special importance for the improvement of cardiac performance by the restoration of the force-frequency relationship in the failing myocardium. The increased expression of the Na⁺-Ca²⁺ exchanger and the modulation of exchanger activity is of great pathophysiological importance for the contractile performance of the failing myocardium and could also provide a direct target for pharmacological interventions in the therapy of heart failure in the future.

	Acknowledgments

 
Experimental work was supported by the Deutsche Forschungsgemeinschaft (M.B., R.H.G.S.). M.B. is a recipient of the Gerhard Hess Program and the Heisenberg Program of the Deutsche Forschungsgemeinschaft. Experiments also were supported by Beiersdorf-Lilly, Hamburg, Germany. This work contains data of the doctoral theses of K.F. and F.S. (University of Cologne, in preparation). We thank K.D. Philipson, PhD, Los Angeles, for providing the cDNA encoding rat heart Na⁺-Ca²⁺ exchanger, Paul Barton, PhD, London, for providing the cDNA encoding human ß-MHC, and Suntory Limited, Osaka, Japan, for providing the cDNA encoding human ANF. The technical assistance of Evelyn Behrendt and Ute Laudenbach-Leschowsky is gratefully acknowledged.

Received December 7, 1995; revision received February 27, 1996; accepted March 4, 1996.

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