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European Journal of Heart Failure 2006 8(7):673-680; doi:10.1016/j.ejheart.2006.01.013
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© 2006 European Society of Cardiology

High intracellular Na+ preserves myocardial function at low heart rates in isolated myocardium from failing hearts

Wolfgang Schillingera,*,1, Nils Teuchera,1, Claus Christiansa, Michael Kohlhaasa, Samuel Sossallaa, Phuc Van Nguyena, Albrecht G. Schmidta, Ortwin Schunckb, Klaus Nebendahlb, Lars S. Maiera, Oliver Zeitza,2 and Gerd Hasenfussa

a Georg-August-Universität Göttingen, Herzzentrum, Kardiologie und Pneumologie, Robert-Koch-Str. 40, 37099 Göttingen, Germany
b Georg-August Universität Göttingen, Zentrale Tierexperimentelle Einrichtung, Göttingen, Germany

* Corresponding author. Tel.: +49 551 39 6349; fax: +49 551 39 9804. E-mail address: schiwolf{at}med.uni-goettingen.de (W. Schillinger).


   Abstract
Top
 Notes
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
We investigated the hypothesis that increased intracellular [Na+]i in heart failure contributes to preservation of SR Ca2+ load which may become particularly evident at slow heart rates.

[Na+]i in SBFI-loaded myocytes from rabbits with pacing-induced heart failure (PHF) was significantly higher at each frequency as compared to Sham-operated animals. Furthermore, PHF rabbits demonstrated reduced SR Ca2+-ATPase protein levels (– 37%, p < 0.04) but unchanged Na+/Ca2+ exchanger protein levels. At 0.25 Hz, isometric force was similar in cardiac trabeculae from PHF rabbits as compared to control (PHF, 3.6±1.3; Sham, 4.4 ± 0.6 mN/mm2). Rapid cooling contractures (RCCs) were unchanged indicating preserved SR Ca2+ load at this frequency. In Sham, isometric twitch force increased with rising frequencies to 29.0 + 2.8 mN/mm2 at 3.0 Hz (p < 0.05) as compared to 0.25 Hz. RCCs showed a parallel increase by 186 ± 47% (p < 0.01). In PHF, frequency-dependent increase in force (15.8 ± 4.7 mN/mm2 at 3.0 Hz) and RCCs (increase by 70 ± 40%) were significantly blunted.

Thus, in PHF in rabbits SR Ca2+ load is preserved at low frequencies despite decreased SR Ca2+-ATPase expression. This may result from [Na+]i-dependent changes in Na+/Ca2+ exchanger activity.

Key Words: Dilated cardiomyopathy • Heart failure • Force–frequency relationship • Sodium • Animal model

Received July 21, 2005; Revised October 28, 2005; Accepted January 25, 2006


   1. Introduction
Top
 Notes
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
During the last decade it has been observed that Ca2+ transients and intracellular Ca2+ cycling are considerably altered in the failing human heart and exaggerated at higher heart rates. Furthermore, it was shown that these changes are the consequence of disturbed sarcoplasmic reticulum (SR) Ca2+ load. It was also realized that this induces impairment of excitation-contraction coupling resulting in blunted or reversed frequency-potentiation of contractile force [1]. Alterations in SR Ca2+ load in heart failure can originate from altered activity and expression of SR Ca2+-ATPase (SERCA) [1,2], increased Ca2+ leak from the SR [3], and reduced Ca2+ availability to SERCA. The latter may be a result of an altered expression and function of the sarcolemmal Na+/Ca2+ exchanger (NCX) as the major alternative mechanism to remove Ca2+ from the cytosol. Increased expression of NCX has been suggested to promote SR Ca2+ depletion by opposing SR Ca2+ uptake [4,5]. However, expression levels of NCX may cover a wide range, and even though expression is unchanged NCX function may be impaired when its driving forces are altered [5,6]. Depending on [Ca2+] and [Na+] at either side of the plasma membrane and on the membrane potential NCX may work in a forward mode promoting Ca2+ efflux and Na+ influx and in a reversed mode promoting opposite ion fluxes. Under physiological conditions, forward mode Na+/Ca2+ exchange eliminates Ca2+ that enters the cell via L-type Ca2+ channels and via reversed mode Na+/Ca2+ exchange during the action potential, thus, maintaining cellular Ca2+ constant during steady-state. As a consequence, any situation affecting NCX function may shift the intracellular Ca2+ balance to a new steady-state with altered SR Ca2+ content [7,8]. Recently, we could demonstrate that [Na+]i was elevated in SBFI-loaded muscle strips from failing as compared to nonfailing human myocardium [9]. Increased [Na+]i promoting Ca2+ influx may contribute to systolic function by increased Ca2+ availability to contractile proteins. On the other hand it may impair diastolic function because of increased diastolic Ca2+ levels.

A variety of animal models has been developed with the aim to study changes in excitation-contraction coupling in heart failure [10]. Regarding Ca2+ elimination processes the use of rabbit myocardium is often considered advantageous owing to its similarity to human hearts [8]. Unlike in small rodents like rats and mice, in which Ca2+ elimination from the cytosol mostly occurs by sarcoplasmic reticulum, NCX becomes more relevant in most mammalian species such as rabbits or humans. Tachycardia pacing-induced heart failure (PHF) in rabbits is characterized by cardiac dilatation and failure without clear phase of functionally compensated hypertrophy. Besides, similarly to what has been reported in the failing human heart this rabbit model demonstrated reduced frequency-dependent potentiation of contractile force [11], abnormal [Ca2+]i homeostasis [12] as well as impaired β-adrenergic recruitment of the contractile reserve [11]. Thus, this model is fairly mimicking changes in excitation-contraction coupling of failing human dilated cardiomyopathy. Accordingly, we used this model to investigate the role of [Na+]i on the force-frequency relation. However, [Na+]i has not been investigated in contracting myocardium from rabbits with PHF. In the present study, we have therefore analyzed [Na+]i at different stimulation rates in rabbits with PHF and found an increase at any stimulation rate similar to the findings in human myocardium. We sought to investigate the hypothesis that the increase in [Na+]i is associated with maintenance of SR Ca2+ load and contractility particularly at slow frequencies when diastolic intervals are long enough for SR Ca2+ uptake. In addition, protein levels of NCX and SERCA were determined.


   2. Methods
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1. Materials
All chemicals and reagents were purchased from commercial suppliers with highest purity grade available.

2.2. Animal model
Chinchilla bastard rabbits (1.5-2.0 kg) were used. The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the U.S. National Institutes of Health (NIH Publication No. 85-23, revised 1985). According to a previously published procedure pacing-induced heart failure (PHF) was induced in rabbits with some minor modifications [11,12]. Briefly, rabbits were anesthetized by injection of S(+)-ketamine (10 mg/kg body weight) and xylazine (5 mg/kg body weight) in the muscle of one thigh. A lateral ear vein was cannulated and atropine (0.025 mg/kg body weight) was administered intravenously. The rabbits were then intubated and mechanical pressure-controlled ventilation at a rate of 40/min was applied. Anesthesia was maintained by continuous infusion of propofol (5-10 mg/kg body weight). Additionally, S(+)-ketamine (2.5 mg/kg body weight) was given every 10-15 min intravenously. Then, the abdominal route with incision of the diaphragm was used. Two pacemaker leads were attached to the apex of the heart and an inactive conventional pacemaker (Quantum III, Intermedics Inc.) was implanted subcutaneously on the pectoralis major muscle. For postoperative analgesia carprofen (5 mg/kg body weight) was given subcutaneously before anesthesia was stopped. After recovering from surgery, the pacemakers were programmed to sustained tachycardia pacing at 400 beats/min by use of an external programmer (RX2000, Intermedics Inc.) with modified software allowing of programming such rapid pacing rates. After 3-5 weeks, fractional shortening as measured by echocardiography had gradually decreased from 0.28±0.03 to 0.13±0.01 (p<0.05). After the fractional shortening was confirmed as being ≤0.15 or alternatively ≤0.20 if ascites or pleural effusion and clinical symptoms of failure (dyspnea, appetite loss, and body weight loss) were present at the same time, rabbits were killed and hearts were quickly removed. Sham operated animals (Sham) underwent all procedures in parallel, but pacemakers remained inactive. In these animals, FS did not significantly change (0.32±0.08 vs. 0.28±0.10). Experiments in Sham were performed 4 weeks after surgery.

2.3. Quantification of NCX and SERCA protein levels
Western blot analyses were performed as previously described [4]. Myocardial samples were taken immediately after explantation, quickly frozen in liquid nitrogen and stored at –80 °C until use. Equal amounts of myocardium were pulverized in a cryo-cap submerged in liquid nitrogen by means of a teflon pastle and solubilized in an ice-cold homogenization buffer (50 mmol/L Na-HEPES, pH 7.4, 50 mmol/L KCl, 1 mmol/L benzamidine, 100 µmol/L EGTA, 1 mmol/L DTT solution containing protein inhibitors: 1 µg/mL Leupeptin, 1 µg/mL Chymostatin, 1 µg/mL Antipain, 1 µg/mL Pepstatin A, and 1 mmol/L PMSF). The suspension was further homogenized by 15 strokes with the pastle. The homogenates were centrifuged at 14,000 rpm at 4 °C. The supernatant was collected, the pellet was resuspended in 50 µL of homogenization buffer and homogenized by 15 strokes with the pastle. The second homogenate was poured to the first supernatant and further homogenized by means of an ultrasound homogenizer (3x10 s). The protein concentrations were determined in triplicate by BCA Protein Assay (Pierce, Rockford, USA). No significant differences were found in the yield of protein per gram wet weight between the different groups. Aliquots of the homogenates were frozen in liquid nitrogen and stored at –80 °C until use. Equal amounts of protein from all samples were subjected to SDS-polyacrylamide gel electrophoresis and electro-blotted to nitrocellulose. The blots were blocked in 5% nonfat milk dissolved in TTBS (20 mmol/L Tris-Cl, pH 7.4, 150 mmol/L NaCl, 0.1% TWEEN), then probed for 2 h with an antibody to NCX (ABR, USA, cat. MA3-926) diluted 1:3000 in TTBS, containing 0.5% nonfat milk, or with antibodies to SERCA (1:10,000) (ABR, USA, clone 2A7-A1), and calsequestrin (1:2000) (ABR, USA, Cat. PAI-913), respectively. Then, the membranes were incubated for one hour with a peroxidase-labeled antibody against rabbit for calsequestrin and against mouse for NCX and SERCA respectively (Amersham Buchler Ltd. UK). Immunoreactive bands were visualized utilizing a chemoluminescence kit (SuperSignal West Pico, Pierce, Rockfort, USA) and exposure to a Kodak X-ray film. Band densities were evaluated using a ChemiImager TM4400 densitometer (Alpha Innotech Corp. CA, USA). Since several blots had to be performed for quantificating each protein in all samples one heart was used as a reference on all blots. NCX and SERCA protein levels were normalized to calsequestrin protein levels to account for differences in connective tissue content. Each individual value represents the mean of two independent determinations. A series of blots was preceded by checking linearity of the assay by plotting different amounts of proteins to corresponding densitometric units.

2.4. Measurement of [Na+]i in isolated myocytes
After cardiectomy, the heart was mounted in a modified Langendorff perfusion setup and perfused with Tyrode I solution (in mmol/L): 137 NaCl, 5.4 KCl, 1.2 Na2HPO4, 1.2 MgSO4, 20 HEPES, 15 glucose, 1 CaCl2) aerated with 100% O2 for 5-8 min. Perfusion was then switched to nominally Ca2+-free Tyrode solution for 12-15 min (30 ml/min) and digestion was performed by perfusion for 12-15 min (10 ml/min) with Tyrode-enzyme solution containing 250 U/ml collagenase type II, and 0.04 mg/ml protease type XIV, 0.025 mmol/L Ca2+, 60 mmol/L taurine, 8 mmol/L D,L-glutamic acid, and 2 mmol/L D,L-carnitine. Digestion was stopped by perfusion with 100 mL Tyrode solution containing 50 µmol/L Ca2+, 2% fatty acid-free type V albumin, and 20 mmol/L 2,3-butanedione monoxime. 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. The final suspension was laid on top of a 6% albumin/M199 medium (1.75 mmol/L Ca2+) supplemented with 5mmol/L D,L-carnitine, 5 mmol/L taurine, 5 mmol/L creatine and antibiotics. Myocytes were counted and plated at a density of 1.4x103 rod-shaped cells/cm2 onto laminin (10 µg/mL)-coated tissue culture dishes. After 2 h, unattached cells were removed by three wash steps. [Na+]i was measured using the Na+-sensitive fluorescent dye benzofuran isophthalate (SBFI; Molecular Probes). Myocytes on laminin-coated culture dishes were incubated at room temperature for 120 min in HEPES solution containing 10 µM SBFI-AM. The loaded myocytes were then washed and incubated in dye-free HEPES solution for 15 min. After incubation, all non-deesterified dye was washed away and measurements could be started. Basic stimulation rate was 0.5 Hz and was continuously increased to 1.0, 2.0, and 3.0 Hz after steady-state values were recorded. Fluorescence was recorded and analyzed as described previously [9]. Briefly, excitation light (75-W mercury xenon, Ushio) was passed alternately through 340- and 380-nm band-pass filters at 240 Hz and focused on the myocyte. The [Na+]i-dependent SBFI fluorescence was directed through a 510-nm band-pass filter and collected by a photomultiplier (IonOptix). After subtraction of autofluorescence, the emitted signals at 340 and 380 nm were divided to obtain the 340/380 ratio. The 340/380 ratios were calibrated by an in vivo procedure as described previously [9].

2.5. Preparation of cardiac trabeculae and registration of mechanical parameters
Immediately after cardiectomy, the hearts were rapidly washed by retrograde perfusion through the aorta with a Krebs-Henseleit solution containing (in mmol/L) 120 NaCl, 5.0 KCl, 2.0 MgSO4, 1.2 NaH2PO4, 20 NaHCO3, 10 glucose, 0.25 CaCl2, and 20 2,3-butanedione monoxime and oxygenated by bubbling with 95% O2-5% CO2. Thin trabeculae or muscle strips were prepared in a dissection chamber, mounted in the muscle chamber and connected to the force gauge (KG4, Scientific Instruments, Heidelberg, FRG). The force transducer signals were amplified and recorded using customized LabView® based software and were normalized to the dimensions and specified in mN/mm2. Muscles were submerged in normal Krebs-Ringer solution (Ca2+ 1.75 mmol/l) at 37 °C and stimulated (25% above threshold voltage; 5 ms duration). Preparations were stretched by a protocol designated to yield a sarcomere length closed to physiological conditions [13]. Force-frequency relation was obtained by recording and measuring steady-state twitch force at each frequency (0.25, 0.5, 1.0, 2.0 and 3.0 Hz) [4,8,9]. Rapid cooling contractures were elicited by a rapid decrease in the temperature of the muscle chamber from 37 °C to 1 °C at each stimulation rate. Rapid cooling causes release of all SR Ca2+ while simultaneously inhibiting Ca2+ transport mechanisms. The amplitude of the resulting contracture is a useful index of the amount of Ca2+ which was stored in the SR at the time of cooling [8].

2.6. Statistical analysis
Data are expressed as mean±SEM. Comparisons of force values (force-frequency and rapid cooling protocols) were performed by repeated measures ANOVA followed by Student-Newman-Keuls' test. Differences between protein levels of the different groups were tested by unpaired t-test. A p-value <0.05 was accepted as statistically significant.


   3. Results
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1. Force-frequency relationship
At slow frequencies (0.25 and 0.5 Hz), basal isometric forces of trabeculae from PHF and Sham were not significantly different. However, relaxation times (RT50% at 0.25 and 0.5 Hz and RT90% at 0.25-1.0 Hz) were significantly prolonged and relaxation velocities (–{Delta}F/{Delta}t at 0.25-1.0 Hz) were significantly slowed down in PHF (Fig. 1, Table 1). Similar to isometric twitch force values, no significant differences in the magnitude of rapid cooling contractures were found between Sham (11.0±2.4 mN/mm2) and PHF (11.6±3.1 mN/mm2, n.s.) at 0.25 Hz (Fig. 2).


Figure 1
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  		Fig. 1 Force-frequency relationship. Panel A: Original data from digital recordings of isometrically contracting ventricular trabeculae from Sham and PHF. A steep frequency-dependent increase in isometric twitch force up to 3.0 Hz was demonstrated in Sham whereas frequency-potentiation of force was flat in PHF. Panel B: Graph showing mean force values in Sham (n=21 trabeculae from 8 animals) and PHF (n=19/6). Basal forces at 0.25 and 0.5 Hz were not significantly different between the groups. *p<0.05 vs. 0.25 Hz, #p<0.05 vs. Sham.

 


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  		Table 1 Force-frequency relationship

 


Figure 2
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  		Fig. 2 Frequency-dependence of rapid cooling contractures. The amplitudes of rapid cooling contractures indicate a frequency-dependent increase in SR Ca2+ load in Sham (n=6/6). This was significantly diminished in PHF (n=4/4). At 0.25 Hz similar amplitudes of contractures were found. *p<0.05 vs. 0.25 Hz, #p<0.05 vs. Sham.

 
With increasing stimulation rates, there was a steady and significant increase in isometric force in Sham by 243±16% at 1.0 Hz, 517±11% at 2.0 Hz, and 560±10% at 3.0 Hz as compared to 0.25 Hz. Frequency-potentiation of force was significantly blunted in PHF. Compared to 0.25 Hz, force increased by 133±43% at 1.0 Hz, 306±35% at 2.0 Hz, and 344±30% at 3.0 Hz in PHF (Fig. 1, Table 1). Concurrently, in Sham the amplitude of cooling contractures significantly increased with rising stimulation rates by 109±23% at 1.0 Hz, 180±41% at 2.0 Hz, and 186±47% at 3.0 Hz indicating frequency-dependent increase in SR Ca2+ load. Rise in rapid cooling contractures (30±17%, 61±23%, and 70±40% at 1.0, 2.0, and 3.0 Hz, respectively) was significantly depressed in PHF (Fig. 2).

3.2. Frequency-dependence of [Na+]i
Fig. 3 summarizes the effect of increasing stimulation rates in SBFI-loaded myocytes in Sham and PHF. Similar to the findings in isolated muscle strips frequency-dependent potentiation of fractional shortening was present in isolated myocytes from Sham-operated animals whereas it was flat or negative in myocytes from rabbits with PHF. SBFI-dependent fluorescence representing [Na+]i did not show cycle related fluctuations in either group of animals. In Sham, [Na+]i was (mmol/L) 4.7±0.4 at 0.5 Hz, 5.6±0.5 at 1.0 Hz, 6.1±0.6 at 2.0 Hz, and 5.7±0.9 at 3.0 Hz. In PHF the corresponding [Na+]i were 8.5±1.3, 9.5±1.4, 11.2±1.3, and 11.8±1.6 mmol/L. Hence, in PHF [Na+]i was significantly increased by a factor of ~2 at any stimulation frequency as compared to Sham.


Figure 3
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  		Fig. 3 Frequency-dependent effects on [Na+]i. Panel A: Dependence of fractional shortening from stimulation frequency in isolated myocytes from Sham and PHF. Original data from digital recordings of typical experiments are shown. Panel B: Original data from digital recordings of [Na+]i-fluorescence (SBFI 10 µM, calibrated) from Sham and PHF. Note that fluorescence was only recorded for a few seconds due to possible effects of bleaching of the indicator. Most of the time, the shutter was closed. Panel C, Graph showing significant elevation of [Na+]i in PHF (n=8 myocytes from 3 animals) compared to Sham (n=9/3) at any stimulation frequency. *p<0.05 vs. 0.5Hz, #p<0.05 vs. Sham.

 
3.3. Protein levels of SERCA and NCX
There was no significant difference in calsequestrin protein levels between Sham and PHF (2384±216 and 2126±430 densitometric units per mg of protein, respectively). SERCA and NCX protein levels were then normalized to calsequestrin in order to ensure that differences in protein levels reflect differences in the myocardial fraction and do not result from differences in non-myocardial tissue like connective tissue or vessels. Relative to calsequestrin, SERCA protein levels were 0.89±0.11 and 0.56±0.08 (–37%, p<0.04) and NCX protein levels were 0.81±0.16 and 0.68±0.12 (n.s.) in Sham and PHF, respectively (Fig. 4).


Figure 4
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  		Fig. 4 Quantification of SERCA and NCX Protein Levels. Panel A: Western-Immunoblots of SERCA, NCX, and Calsequestrin (CALSQ) in Sham (n=6) and PHF (n=5). Molecular weights determined by marker proteins are indicated in kDa. Panel B: Bar graphs showing mean protein levels of NCX and SERCA normalized to calsequestrin. #p<0.04 vs. Sham.

 

   4. Discussion
Top
 Notes
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The main findings of this study are that (1) in pacing-induced heart failure in rabbits [Na+]i is increased at any stimulation frequency as compared to sham-operated animals. (2) SERCA protein levels are decreased whereas NCX protein levels are not significantly altered. (3) At low frequencies, contractile force and SR Ca2+ load are preserved and relaxation is prolonged. (4) At high heart rates, contractile force is reduced and relaxation unaltered. We conclude that increased [Na+]i at low heart rates preserves systolic function but prolongs relaxation because of increased Ca2+ influx. At high heart rates with shorter action potential the effect of high [Na+]i disappears.

Recently, extensive work has elucidated the role of SERCA expression and function in end-stage heart failure. Decreased expression and/or function seem to be a consistent finding in the failing human and animal heart. Downregulation of SERCA is associated with impaired Ca2+ transients and contractile dysfunction caused by decreased SR Ca2+ load. At fast heart rates impaired SR function becomes particularly evident [1]. Moreover, SR Ca2+ load, Ca2+ transients, and frequency-dependent force-potentiation could be restored by gene transfer of SERCA [2]. Consistently, the present work shows reduced SERCA protein levels in rabbits with PHF along with reduced contractile twitch force and SR Ca2+ load as measured by the amplitude of rapid cooling contractures at fast heart rates.

Previous studies have investigated NCX expression and function in failing myocardium from several species including humans. Increased expression was a frequent finding, but unchanged expression was also found. Besides, wide inter-individual differences may be found in the same species [4-6]. In the present study, no significant changes were found with respect to NCX protein levels.

During relaxation there is dynamic competition between NCX and SERCA for cytoplasmic Ca2+. Thus, SR Ca2+ load in steady state contracting myocytes depends on the relative contribution of both competitors. Assuming that protein levels of NCX and SERCA reflected activity and forward mode Na+/Ca2+ exchange was the predominant working mode of NCX in rabbit hearts it might have been expected that contractile force and SR Ca2+ load were reduced in rabbits with PHF throughout the whole range of frequencies. However, at slow stimulation frequencies SR Ca2+ load and isometric twitch force were preserved. Like in the present work preservation of contractile force at slow heart rates has also been found recently in a subgroup of human heart failure patients with preserved NCX and decreased SERCA protein levels [4]. Moreover, consistent with our findings peak systolic [Ca2+] and SR Ca2+ load have been shown to be similar in failing compared to nonfailing human hearts at slow heart rates [4,9,14].

It is obvious that protein levels alone are not sufficient for appreciating the functional role of NCX for SR Ca2+ load. Thus, concomitant changes have to be taken into account. The driving force of NCX (ENCX) is a function of the trans-sarcolemmal electrochemical potential differences of Na+ and Ca2+ and of the sarcolemmal membrane potential [5,7,8]. Increase in [Na+]i stimulates reversed mode ENCX during the action potential and depress forward mode ENCX during diastole. Bers calculated that an increase in [Na+]i from 9 to 12mmol/L could shift the reversal potential of NCX by –30 mV [8]. Therefore, increase in [Na+]i favors Ca2+ loading of the cytoplasm and of the SR [5,7,8]. Previous studies in mice that have physiologically higher [Na]i than rabbits and humans [8] showed an increased SR Ca2+ content after transgenic overexpression of NCX [15] whereas sole adenoviral overexpression of NCX in rabbit myocytes with unaltered [Na]i resulted in decreased SR Ca2+ content [16]. Recently, in humans and animals [Na+]i has been shown to be increased in heart failure [9,17,18]. This was suggested to originate from reduced expression and activity of Na+/K+-ATPase in human heart failure [6] and from increased Na+ influx through Na+ channels [17] or via the sarcolemmal Na+/H+ exchanger [18] in animal models.

In the present work that investigated rabbits with PHF, [Na+]i was increased at each stimulation frequency. In view of this finding we interpret the preservation of SR Ca2+ load and isometric twitch force at low heart rates as a result of [Na+]i-dependent changes in NCX activity. This is associated with a reduced velocity of relaxation and a prolonged relaxation time at slow heart rates. Consistently, a study of Yao et al. using the same model showed a prolongation of the terminal phase of the Ca2+ transient [12]. We therefore conclude that the observed effects at slow frequencies with prolonged action potential resulted from increased reversed mode Na+/Ca2+ exchange, thereby compensating for decreased SERCA protein levels.

In conclusion, in isolated trabeculae from rabbits with PHF SR Ca2+ load and contractility is preserved at slow stimulation frequencies at the expense of prolonged relaxation. This may be the consequence of an increased Ca2+ influx through NCX by substantially elevated [Na+]i. However, with increasing stimulation frequencies and decrease of the action potential duration [Na+]i-dependent changes in NCX activity are not sufficient to compensate for reduced SERCA expression resulting in impaired frequency-dependent potentiation of SR Ca2+ load and contractile force. These findings emphasize that PHF in rabbits is an appropriate model for investigating excitation-contraction coupling in failing human dilated cardiomyopathy. The extrapolation of our data to the in vivo situation of the failing heart may be limited by the fact that the frequency range in our study was below the physiological rates of conscious rabbits. However, in view of this limitation the data provide additional evidence for the necessity of rate control in heart failure.


   Acknowledgements
 
This work was supported by the Georg-August-Universität Göttingen, Forschungsförderungsprogramm to Wolfgang Schillinger and by the Deutsche Forschungsgemeinschaft, Sonderforschungsbereich Transregio 2 to Gerd Hasenfuss, Emmy-Noether-Programm MA 1982/1-2, 1-4 and GlaxoSmithKline Research Foundation to Lars Maier, and Graduiertenkolleg 521 to Nils Teucher. We gratefully acknowledge the expert assistance of Jessica Spitalieri, Michael Kothe, Melanie Bernhardt, and Corinna Fitzner.


   Notes
Top
 Notes
Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
1 Both authors contributed equally to this work. Back

2 Current address: Universitätsklinikum Hamburg-Eppendorf, Klinik und Poliklinik für Augenheilkunde, Hamburg, Germany. Back


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

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W. Schillinger, C. Christians, S. Sossalla, N. Teucher, P. Nguyen Van, H. Kogler, O. Zeitz, and G. Hasenfuss
{alpha}1-adrenergic stress induces downregulation of Na+/Ca2+ exchanger in myocardial preparations from rabbits at physiological preload
Eur J Heart Fail, April 1, 2007; 9(4): 329 - 335.
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