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Cardiovascular Research Advance Access originally published online on January 17, 2008
Cardiovascular Research 2008 78(1):71-78; doi:10.1093/cvr/cvn013
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Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2008. For permissions please email: journals.permissions@oxfordjournals.org.

Altered Na+/Ca2+-exchanger activity due to downregulation of Na+/K+-ATPase {alpha}2-isoform in heart failure{dagger}

Fredrik Swift1,2,*, Jon Arne Kro Birkeland1,2, Nils Tovsrud1,2, Ulla H. Enger1,2, Jan Magnus Aronsen1,2, William E. Louch1,2, Ivar Sjaastad1,2,3 and Ole M. Sejersted1,2

1 Institute for Experimental Medical Research, Ullevaal University Hospital, Kirkeveien 166, N-0407 Oslo, Norway
2 Center for Heart Failure Research, Faculty of Medicine, University of Oslo, Oslo, Norway
3 Department of Cardiology, Ullevaal University Hospital, Oslo, Norway

* Corresponding author. Tel: +47 23016800; fax: +47 23016799. E-mail address: fredrik.swift{at}medisin.uio.no

Received 15 October 2007; revised 8 January 2008; accepted 11 January 2008

Time for primary review: 16 days


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
Aims: The Na+/K+-ATPase (NKA) {alpha}2-isoform is preferentially located in the t-tubules of cardiomyocytes and is functionally coupled to the Na+/Ca2+-exchanger (NCX) and Ca2+ regulation through intracellular Na+ concentration ([Na+]i). We hypothesized that downregulation of the NKA {alpha}2-isoform during congestive heart failure (CHF) disturbs the link between Na+ and Ca2+, and thus the control of cardiomyocyte contraction.

Methods and results: NKA isoform and t-tubule distributions were studied using immunocytochemistry, confocal and electron microscopy in a post-infarction rat model of CHF. Sham-operated rats served as controls. NKA and NCX currents (INKA and INCX) were measured and {alpha}2-isoform current (INKA,{alpha}2) was separated from total INKA using 0.3 µM ouabain. Detubulation of cardiomyocytes was performed to assess the presence of {alpha}2-isoforms in the t-tubules. In CHF, the t-tubule network had a disorganized appearance in both isolated cardiomyocytes and fixed tissue. This was associated with altered expression patterns of NKA {alpha}1- and {alpha}2-isoforms. INKA,{alpha}2 density was reduced by 78% in CHF, in agreement with decreased protein expression (74%). When INKA,{alpha}2 was blocked in Sham cardiomyocytes, contractile parameters converged with those observed in CHF. In Sham, abrupt activation of INKA led to a decrease in INCX, presumably due to local depletion of [Na+]i in the vicinity of NCX. This decrease was smaller when the {alpha}2-isoform was downregulated (CHF) or inhibited (ouabain), indicating that the {alpha}2-isoform is necessary to modulate local [Na+]i close to NCX.

Conclusion: Downregulation of the {alpha}2-isoform causes attenuated control of NCX activity in CHF, reducing its capability to extrude Ca2+ from cardiomyocytes.

KEYWORDS Heart failure; Na/K-ATPase; Na/Ca-exchanger; e–c coupling; Contractile function


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 Funding
 References
 
The Na+/Ca2+-exchanger (NCX) is the main extrusion pathway of Ca2+ from cardiomyocytes through forward mode exchange.1 However, in the reverse mode, it can bring Ca2+ into the cardiomyocyte, contributing to an increased Ca2+ load of the sarcoplasmic reticulum (SR). Also, although still controversial, it could play a role in trigging Ca2+ release from the SR.2 Intracellular Na+ concentration ([Na+]i) is a main regulator of exchange mode and activity of the NCX. Since diffusion of Na+ in the cytosol seems to be restricted,3–7 it is possible that accumulation and depletion of Na+ can occur in various localized submembrane ‘pockets’ (‘fuzzy space’8,9) during the cardiac cycle. Changes in Na+ concentrations in a submembrane space ([Na+]ss) may affect the function of nearby NCX.10

The Na+/K+-ATPase (NKA) is an important determinant of [Na+]i. Rat cardiomyocytes have {alpha}1- and {alpha}2-isoforms of the NKA. Although both seem to be functionally coupled to NCX,11,12 it has been suggested that the {alpha}1-isoform plays a more ‘housekeeping’ role, whereas the {alpha}2-isoform is selectively involved in Ca2+ regulation.13 A likely explanation for this is that, provided slow diffusion of Na+ in the cytosol, a subgroup of NKAs may control local [Na+]ss in the vicinity of NCX. In cultured astrocytes it was demonstrated that the NKA {alpha}2-isoform colocalizes with NCX.14 In a recent study in cardiomyocytes, we showed that even though the {alpha}2-isoform of the NKA constitutes only ~10% of all the NKA in the cardiomyocyte, selective inhibition of the {alpha}2-isoform produced a positive inotropic response.10 However, this effect was not mediated through a global rise in [Na+]i, but through local regulation of NCX,10 probably through higher [Na+]ss. Such a role for the {alpha}2-isoform has been suggested, but has not been clearly demonstrated.13,15,16

Both the {alpha}2-isoform and NCX are located in the t-tubules.10,17 T-tubules are invaginations of the sarcolemma which form a tortuous network in cardiomyocytes and in some regions come very close to the SR (dyads), so that proteins of the two membrane systems involved in excitation–contraction coupling can interact. Multiple lines of evidence suggest alterations in the t-tubule structure during heart failure. It has been reported that these changes in the t-tubules are associated with less synchronous Ca2+ release in various models of heart failure.18,19 This impairment of Ca2+ handling could potentially result from altered coupling between the {alpha}2-isoform and NCX during t-tubule remodelling. Supporting this idea it has been shown that the protein level of the {alpha}2-isoform was reduced in a post-infarction model of heart failure.20 The functional consequences of altered {alpha}2-isoform levels and expression patterns in heart failure have not been addressed.

The aim of the present study was to determine the expression pattern of the {alpha}2-isoform in a post-infarction rat model of congestive heart failure (CHF) and to identify the functional consequences of NKA {alpha}2-isoform downregulation. We hypothesized that disorganization of t-tubules associated with downregulation of the NKA {alpha}2-isoform in CHF disturbs the link between Na+ and Ca2+, and thus control of cardiomyocyte contraction.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 Funding
 References
 
2.1 Animal care
The investigation conforms with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH publication No. 85-23, revised 1996). Two animals were kept in each cage and housed in a temperature-regulated room with a 12-h day/12-h night cycling, and had access to food and water ad libitum. Male Wistar rats (Møllegaard, Skensved, Denmark) ~70 days old, weighing ~300 g were anaesthetized with 68% N2O, 29% O2 and 1.5–2% isofluran (Abbott Scandinavia, Solna, Sweden), and ventilated [40/min, inspiration : expiration ratio = 1 : 2, pressure 20/5 mmH2O (max/mean)] on a respirator (Zoovent, Triumph Technical Services, Milton Keynes, UK). Myocardial infarction was induced by ligature of the left coronary artery. After 6 weeks, CHF was attested by increased left ventricular end diastolic pressure (LVEDP ≥ 15 mmHg) and lung weight (LW), as earlier described.21 Sham operated animals served as controls.

2.2 Cardiomyocyte preparation
Single cardiomyocytes were isolated enzymatically as previously described.22 Ca2+ was not reintroduced to cardiomyocytes used for immunocytochemistry.

2.3 Evaluation of t-tubules
The t-tubule network in isolated cardiomyocytes was examined by Di-8-ANEPPS staining, as previously described.23 Confocal images (Zeiss LSM510, x63 water immersion objective, 2048 x 600 pix) were deconvolved using Huygens Essential software (SVI, The Netherlands), and resized to 250 x 73 pix using ImageJ software (http://rsb.info.nih.gov/ij/) before analysis. The one-dimensional power spectrum of each image was measured as the magnitude of the fast Fourier transform computed by the FFT tool in Matlab (MathWorks, Natick, MA, USA). The mean power spectrum was interpolated due to unequal pixel spacing in separate images, and then plotted as a function of spatial frequency in the longitudinal direction of the cell.

2.4 Electron microscopy
Hearts (two CHF, three Sham) were fixed by perfusion through the abdominal aorta as described,24 using 4% formaldehyde and 0.1% glutaraldehyde. Tissue samples were taken from the left ventricle posterior free wall in the non-infarcted area, subjected to freeze substitution and infiltrated in Lowicryl as described.25 Ultra-thin (80–100 nm) longitudinal sections (four CHF, five Sham) were cut and mounted onto nickel grids for blinded examination in the electron microscope. Images were acquired randomly in intact areas of the sections. T-tubule regions were defined as apparent t-tubules or locations where one would expect to see a t-tubule (within or close to a Z line where mitochondria are apposed). T-tubule regions from 25 000x magnification micrographs were categorized into five groups based on their morphology (blinded). Group 1: intact t-tubules with associated SR; group 2: intact t-tubules without apparent associated SR; group 3: scattered and dispersed t-tubules with or without associated SR; group 4: no apparent t-tubule; group 5: non-classifiable.

2.5 Immunocytochemistry
Isolated cardiomyocytes were fixed and immunocytochemistry was performed as described earlier.10 Primary antibodies used were: anti-{alpha}1 (cat#05–369 Upstate, 1 : 100), anti-{alpha}2 McHERED (courtesy of Alicia McDonough, 1 : 100) or anti-{alpha}2 (cat#07–674 Upstate, 1 : 200). One-dimensional power spectrums were generated from deconvolved confocal images as described above for t-tubules.

2.6 Immunoblot analysis
Western blot analyses were performed on protein homogenates as previously described10 with anti-{alpha}1 (1 : 2500) and anti-{alpha}2 McHERED (1 : 3000) antibodies.

2.7 Electrophysiological recordings
Whole-cell voltage clamp protocols modified from5 were used to measure NKA currents (INKA) and NCX currents (INCX). Briefly, cardiomyocytes were superfused (37°C) with solution I containing (mM): NaCl 147, MgCl2 2, EGTA 0.1, D-glucose 5.5, HEPES 5, BaCl2 2, nicardipine 0.001, pH 7.40. INKA and INCX were elicited at a holding potential of –50 mV by adding 5.4 mM KCl and 2 mM CaCl2 to solution I, respectively. Voltage ramp protocols were applied before and during activation of the INKA. Cardiomyocytes were depolarized to +70 mV for 50 ms, then hyperpolarized to –120 mV (dV/dt = 380 mV/s), then stepped to –50 mV. The INKA–voltage relationship was measured during the hyperpolarizing phase of the ramp pulse. INKA activated at each voltage was calculated by subtracting the current recorded at baseline from the current recorded during activation of NKA. To assess the {alpha}2-isoform related INKA (INKA,{alpha}2), we used a low concentration of ouabain (0.3 µM) previously demonstrated to selectively inhibit the {alpha}2-isoform, without blocking the {alpha}1-isoform.10 The intracellular solution was clamped at high [Na+]i using wide bore pipettes (~1 M{Omega}) filled with (mM): NaOH 40, sodium phosphocreatine 5, HEPES 10, TEA-Cl 20, L-aspartic acid 42, EGTA 42, CaCl2 29.7, MgATP 10, pH 7.20. Detubulation was performed as previously described.10

2.8 Contractions
Contractions were recorded in field stimulated cardiomyocytes in (mM): NaCl 140, D-glucose 5.5, KCl 5.4, HEPES 5, CaCl2 1.8, MgCl2 0.5, NaH2PO4 0.4, pH 7.40 as described earlier.10

2.9 Statistics
Values are expressed as means ± SEM. Comparisons between means were made using Student’s t-test (equal variances, two-sided) and comparisons of proportions by a z-test with Yates correction. Comparisons of counts were made by a normal approximation to the difference between two Poisson distributed variables.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 Funding
 References
 
3.1 Animal characteristics
All CHF rats included in the study had haemodynamic signs of heart failure attested by increased LVEDP (Table 1). They also had signs of congestion and hypertrophy, reflected in increased lung weight/body weight and heart weight/body weight ratios. Tachypnea, increased size of the left atrium and pleural effusion were also observed in the CHF group (data not shown).


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  		Table 1 Animal characteristics

 
3.2 Disorganized appearance of t-tubules in congestive heart failure
The t-tubule network of cardiomyocytes from CHF hearts had a disorganized appearance compared with Sham: less continuous staining in the transverse direction and more apparent t-tubules in the longitudinal direction (Figure 1A). Also shown are scans of smaller areas within the cell interior and their respective binary images (threshold at mean intensity). Spectral analyses were performed on these images (Figure 1A,b). The mean one-dimensional power spectrum (Figure 1B) from CHF and Sham showed increased power at regular intervals (spatial frequency of 0.54 µm–1 equal to a distance of 1.85 µm), corresponding to t-tubules in the transverse direction (P < 0.05). However, in the CHF group, the power was lower at these regular intervals reflecting altered organization of t-tubules. Moreover, the power was increased between peaks in the power spectrum of CHF, suggesting increased occurrence of t-tubule segments in the longitudinal direction (P < 0.05).


Figure 1
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  		Figure 1 Altered t-tubule morphology in isolated congestive heart failure (CHF) cardiomyocytes. (A) Representative confocal images of cardiomyocytes stained with di-8-ANEPPS. Images of whole cells (a) and cell sections (b) were deconvolved before analysis. Mean pixel intensity values measured in images showed in (b) served as threshold value to generate binary images (c). (B) Spatial frequency analysis. Mean one-dimensional power spectra of images shown in 1A, b (CHF, n = 21; Sham, n = 19; *P < 0.05).

 
To verify that disorganization of t-tubules was not simply due to CHF cells being less tolerant to processing (e.g. cell isolation), we also evaluated the t-tubules in electron micrographs of fixed tissue (Figure 2). The overall structure at x7 900 magnification appeared less organized in the CHF specimens compared with Sham (Figure 2A), but this was not further quantified. A total of 346 t-tubule regions from CHF hearts and 454 regions from Sham hearts were identified in the 25 000x magnification images (area of image ~11 µm2, total of 78 images from CHF, 88 images from Sham hearts). The calculated t-tubule densities in each group are presented in Figure 2B. Overall t-tubule density was 40.0 t-tubules/µm2 in CHF and 46.6 t-tubules/µm2 in Sham (P < 0.01, Poisson distributed variables). The t-tubule regions were categorized into five groups (see Methods): group 1 (279; CHF 84, Sham 195), group 2 (104; CHF 53, Sham 51), group 3 (336; CHF 166, Sham 170), group 4 (53; CHF 27, Sham 26), group 5 (28; CHF 16, Sham 12). There were relatively less intact t-tubule regions (group 1) in CHF than Sham hearts (P < 0.05, z-test). No differences were observed between CHF and Sham in the remaining groups. However, for groups 2, 3, and 4 pooled, there were more t-tubules in these categories in the CHF group than in the Sham group (P < 0.05, z-test), indicating that the t-tubule morphology had changed from that of group 1 into that of one of the other groups.


Figure 2
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  		Figure 2 Altered t-tubule morphology in intact congestive heart failure (CHF) heart tissue. (A) Representative electron micrographs from Sham and CHF hearts at two magnifications. Scale bar = 1 µm. (B) T-tubule density. T-tubules in x25 000 images were categorized into five groups based on their morphology (representative examples in bottom panel) and counted. Bar chart shows calculated t-tubule density based on the total area of the micrographs.

 
3.3 Altered expression of {alpha}1- and {alpha}2-isoforms in congestive heart failure
Figure 3A shows deconvolved confocal images of cardiomyocytes labelled with antibodies against {alpha}1- and {alpha}2-isoforms of NKA. In Sham, the {alpha}1-isoform was apparent in the surface membrane, the intercalated discs and in both transverse and longitudinal t-tubules. This distribution was also seen in CHF, but appeared to be more disorganized. FFT-analyses of {alpha}1-isoform distribution, shown in Figure 3B, indicated similar power spectrum as from di-8-ANEPPS stained cells showed in Figure 1A. This indicates that altered {alpha}1-isoform distribution might be due to disorganization of t-tubules. However, in the {alpha}1-isoform power spectrum, a high power was observed for spatial frequencies corresponding to the spaces between the transverse t-tubule segments in both CHF and Sham. This probably resulted from differences in the labelling protocols. The distribution of {alpha}2-isoforms was assessed using two different antibodies. Both showed similar results, with labelling of the intercalated discs, but little in the surface membrane. In Sham and CHF, the transverse t-tubules were labelled, with weaker labelling of the longitudinal t-tubules. FFT-analyses of images labelled with both anti-{alpha}2 antibodies showed lower power of labelling in the transverse t-tubules, consistent with the distribution of t-tubules shown in Figure 1A. Immunoblots (Figure 3C) showed a ~19% lower expression of {alpha}1-isoform and a ~74% lower expression of {alpha}2-isoform in CHF.


Figure 3
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  		Figure 3 Altered subcellular expression patterns of Na+/K+-ATPase (NKA) in congestive heart failure (CHF). (A) Representative confocal images of cardiomyocytes labelled with NKA antibodies ({alpha}1, {alpha}2-Upstate, and {alpha}2-McHERED as indicated), with magnifications from the cell interior. Scale bar = 10 µm. (B) Spatial frequency analyses. Mean one-dimensional power spectra of the cell interior images for each antibody used ({alpha}1, {alpha}2-Upstate, and {alpha}2-McHERED from left to right). n = 10–13 images in each group. (C) Protein expression measured by immunoblots in homogenates (top panel). Mean data are presented as bars (n = 6, *P < 0.05, **P < 0.01 CHF vs. Sham).

 
3.4 Impaired function of {alpha}2-isoforms in congestive heart failure
The function of the NKA {alpha}2-isoform was assessed using 0.3 µM ouabain. Since the {alpha}2-isoforms are preferentially located in the t-tubules, we also used detubulation as a method to isolate the {alpha}2-isoforms. Successful detubulation was verified in each batch of cells by staining with di-8-ANEPPS (Figure 4), and was attested by the lack of staining in the t-tubules. Figure 4 also shows cell capacitance measured as the integrated capacitive current during a 10 mV hyperpolarizing step. Reduction of capacitance after detubulation reflects the amount of membrane uncoupled from the sarcolemma by the detubulation. The reduction was similar in Sham and CHF (57.5 pF and 57.6 pF respectively). Capacitance was also increased in CHF compared with Sham reflecting hypertrophy of CHF cardiomyocytes (P < 0.01). A representative current recording from a Sham cell is presented in Figure 5A. Peak INKA density measured right after addition of K+ was lower by 0.75 pA/pF (~26%) in CHF cells compared with Sham cells (P < 0.01). Ouabain (0.3 µM) caused a mean reduction in INKA density in Sham cells of 0.24 pA/pF (P < 0.01, Figures 5B and C). This reduction in current was attributed to the INKA,{alpha}2. The effect of ouabain was small in CHF (0.05 pA/pF, P < 0.05, Figure 5C), indicating loss of {alpha}2-isoform function. After detubulation of Sham cells, a smaller effect of ouabain and hence a lower INKA,{alpha}2 was measured, confirming that the {alpha}2-isoform is preferentially located in the t-tubules. No change was observed in INKA between intact and detubulated CHF cells. As shown in Figure 5D, no significant change in voltage dependence was observed for the {alpha}1-isoform in CHF compared with Sham. However, the calculated INKA,{alpha}2–voltage relationship was significantly different in the CHF cells compared with Sham. At potentials negative to –40 mV, INKA was lower, and almost abolished at normal resting potential (–70 mV).


Figure 4
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  		Figure 4 Detubulation of cardiomyocytes. (A) Representative confocal images of cardiomyocytes stained with di-8-ANEPPS. Successful detubulation was attested by the absence of staining of the t-tubules. (B) Bar chart shows capacitance in control and detubulated cardiomyocytes included in the analysis (n = 11–18 cells, *P < 0.01).

 


Figure 5
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  		Figure 5 Reduced Na+/K+-ATPase (NKA) {alpha}2-isoform function in congestive heart failure (CHF). (A) Representative current recording from a Sham cell during applications of 5.4 mM KCl to elicit INKA and {alpha}2-isoform selective inhibition by 0.3 µM ouabain. Voltage ramps are indicated by letters (a–d). (B) NKA current densities recorded in Sham and CHF cells during the protocol presented above. Mean data are presented with square symbols (CHF, n = 11; Sham, n = 18). (C) Mean {alpha}2-isoform specific currents expressed as reduction in total INKA after application of 0.3 µM ouabain (n = 11–18 cells, *P < 0.05). (D) Current–voltage relationships for {alpha}1- and {alpha}2-isoforms recorded during voltage ramps a–d (CHF, n = 6; Sham n = 12, *P < 0.05).

 
3.5 Converging contractile properties in Sham and congestive heart failure during {alpha}2-isoform inhibition
Representative examples of contractions measured in field stimulated cells before and after 9 min of exposure to 0.3 µM ouabain, are presented in Figure 6A. The effect of {alpha}2-isoform inhibition on contractions increased with time, consistent with gradual accumulation of Ca2+ in the SR. Mean data are presented in Figure 6B. Contractility is expressed as fractional shortening/time to peak (FS/TTP). Values were not different between CHF and Sham at any time point (power > 0.8, {alpha} < 0.05). However, the increase in FS/TTP was lower in CHF than in Sham at all time points after 2 min, and reached 15 ± 5% in CHF and 31 ± 5% in Sham after 9 min (P < 0.05). This could be attributed to loss of {alpha}2-isoforms in CHF.


Figure 6
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  		Figure 6 Converging contractile parameters in Sham and congestive heart failure (CHF) during inhibition of the {alpha}2-isoform. (A) Representative contraction recordings in Sham and CHF in control (solid lines) and after 9 min exposure to 0.3 mM ouabain (dashed lines). (B) Mean fractional shortening (left) and fractional shortening/time to peak (FS/TTP) measurements (right) in Sham (blue) and CHF (red) cardiomyocytes during application of 0.3 µM ouabain (CHF, n = 11; Sham, n = 9, *P < 0.05).

 
3.6 Disrupted cross-talk between Na+/K+-ATPase and Na+/Ca2+-exchanger after {alpha}2-isoform downregulation and inhibition
To assess cross-talk between NKA and NCX, INCX was measured during brief periods of Ca2+ addition prior to and during activation of INKA. A representative current recording from a Sham cell is presented in Figure 7A. In order to verify the stability of the cell prior to activating INKA, two consecutive INCX (NCX1 and NCX2) were measured. NCX1 served as control (Sham; 0.84 ± 0.05 pA/pF, CHF; 0.69 ± 0.05 pA/pF, Sham ouabain; 0.72 ± 0.09 pA/pF, CHF ouabain; 0.75 ± 0.04 pA/pF, not significant). Following activation of NKA, INKA decreased from an initial peak as intracellular Na+ was pumped out of the cell and was accompanied by a decrease of INCX from control. Since global [Na+]i was clamped using low resistance pipettes, the decrease of both INKA and INCX reflects reduction of [Na+] in the subsarcolemmal space. When INKA reached a new steady state, INCX was monitored again (NCX5), and the measured currents are presented relative to NCX1 in Figure 7B. The reduction in current from NCX1 to NCX5 was smaller in CHF than in Sham, indicating less reduction in [Na+]ss during pump activation in the CHF group. To isolate the contribution of the {alpha}2-isoform to the fall in NCX current, the same experiment was conducted in presence of 0.3 µM ouabain. In Sham, the reduction in INCX became similar to that observed in CHF. Furthermore, ouabain had no significant effect in the CHF group, consistent with a low expression of the {alpha}2-isoform. These results indicate that {alpha}2-isoforms are necessary to modulate [Na+]ss in the vicinity of NCX. Figure 7C shows the reduction in INKA (from peak to steady state) as a function of the reduction in NCX5. In Sham, this relationship was linear (correlation coefficient r = 0.77, P < 0.02), suggesting a close coupling between NKA and NCX. When the {alpha}2-isoform was downregulated (CHF) or inhibited (ouabain), this coupling was abolished (CHF: r = 0.39, CHF ouabain: r = 0.45, Sham ouabain: r = 0.21, n = 8–9, P > 0.2).


Figure 7
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  		Figure 7 Altered cross-talk between the Na+/K+-ATPase (NKA) {alpha}2-isoform and the Na+/Ca2+-exchanger (NCX) in congestive heart failure (CHF). (A) Representative current recording from a Sham cell during activation of INCX (NCX1–8) and INKA. (B) Mean values of the NCX5 current (at steady state during NKA activation) relative to NCX1 (n = 8–10 cells, *P < 0.05). (C) The reduction of INCX (NCX1–NCX5) in Sham was linearly dependent on the reduction in NKA (from initial value to steady state). This relationship was abolished in the CHF, Sham ouabain, and CHF ouabain groups.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
Funding
 References
 
In this study, we show that the t-tubule network was disorganized in cardiomyocytes from rat post-infarction CHF hearts. This was associated with altered expression patterns of NKA {alpha}1- and {alpha}2-isoforms. Downregulation of protein level and function of the {alpha}2-isoform in t-tubules of cardiomyocytes from CHF hearts caused attenuated control of NCX activity. This might constitute a molecular basis for the altered contractile properties observed in CHF.

4.1 Methodological considerations
Some methodological aspects should be addressed. First, under similar conditions in normal cells, we determined by establishing a concentration–response curve, that 0.3 µM ouabain blocked ~94% of {alpha}2-isoforms, and only ~1% of {alpha}1-isoform.10 This concentration was therefore used to selectively block the {alpha}2-isoform in the present study in both CHF and Sham cells, assuming unaltered ouabain affinity.

Second, cardiomyocytes were detubulated using formamide to evaluate the fraction of NKA in the t-tubules. Formamide could potentially cause redistribution of NKA isoforms, or have other direct effects on protein properties. However, exposure to formamide (detubulation) had no effect on CHF cells with regard to reduction of INKA by ouabain (Figure 5C), indicating that exposure to formamide did not have direct effects on NKA. Moreover, the detubulation technique has been validated in several papers.17,26–28 In particular, no effects on cytoskeleton or protein properties were found in atrial cells, which do not have t-tubules.28

4.2 T-tubule disorganization in congestive heart failure
As shown in isolated cells stained with a membrane marker (di-8-ANEPPS), the t-tubule network in post-infarction CHF was altered. Studies of heart failure in spontaneously hypertensive rats,19 mouse post-infarction CHF18 and canine tachycardia-induced heart failure29 are in agreement with this conclusion. However, cell damage could result from isolation procedures, and CHF cells could be more fragile and prone to damage. Therefore, we verified our results using electron microscopy of fixed heart tissue, showing that in addition to a decrease in the t-tubule density, there were fewer regions where t-tubules were associated with the SR. This supports the findings that remodelled t-tubules in heart failure constitute a structural basis for orphaned RyRs, leading to ‘Ca2+ instability’.18,19

4.3 Redistribution of Na+/K+-ATPase in congestive heart failure
The intense labelling of NKA {alpha}1-isoforms observed in CHF supports the immunoblot data, showing only a minor reduction in {alpha}1-isoform protein (Figure 3). This reduction might contribute to the ~26% reduction in INKA in CHF. However, as pointed out by Semb et al.,20 it is important to take into account that the CHF cells are hypertrophied, so that any normalization to total protein (as in western blots) or to cell size will be reflected in a reduced expression. Hence, it may well be that the number of NKA {alpha}1-isoform copies per cell may not be different in CHF compared with Sham. The {alpha}2-isoform expression was reduced by ~74% in CHF on the protein level. The subcellular distribution of both {alpha}1- and {alpha}2-isoforms followed the distribution pattern of the t-tubules in CHF, suggesting that they were still targeted to the sarcolemma. However, in the {alpha}1-isoform FFT-analysis (Figure 3B) the relatively high power for spatial frequencies corresponding to the spaces between the transverse t-tubules indicates abundant localization of {alpha}1-isoforms in longitudinal t-tubule segments. This is not reconcilable with the relatively low power for the corresponding spatial frequencies in experiments where solely the sarcolemma was stained (Figure 1B). An explanation for this could be higher background fluorescence in the {alpha}1-isoform labelling experiments. These were performed on fixed, permeabilized cells whereas the di-8-ANEPPS experiments were performed on live cells. Therefore, differences in power spectrums from different labelling protocols should be interpreted with care.

The detubulation experiments (Figure 5C) showed that since the effect of ouabain was lowered by ~50% in Sham cells after detubulation, and that t-tubules constitute ~30% of the total sarcolemma, the {alpha}2-isoforms in Sham cells were preferentially located in the t-tubules. However, because detubulation was probably not 100% complete, the fraction of {alpha}2-isoforms in the t-tubules was certainly underestimated. This is in agreement with our previous observations in normal cells.10

4.4 Functional consequences of downregulation of {alpha}2-isoform function in congestive heart failure
INKA,{alpha}2 density was reduced by 78% in CHF (Figure 5C). This fits with the protein levels measured by immunoblots (74%, Figure 3C). Thus, INKA,{alpha}2 seems directly dependent on protein levels. This is in line with investigations showing that phospholemman does not regulate the {alpha}2-isoform.15,30

The current–voltage relationship of the {alpha}2-isoforms in CHF (Figure 5D) shows that at normal resting potentials, the INKA,{alpha}2 was not detectable, in agreement with the reduced protein levels. However, the INKA,{alpha}2 reached Sham levels at positive potentials indicating a potentiated INKA,{alpha}2 voltage dependence in CHF cells. We have no explanation for this. We speculate that at positive potentials, the INKA,{alpha}2 in CHF has an increased affinity for Na+. The proposed implication of the steep voltage dependence of the {alpha}2-isoform in both Sham and CHF is that the NKA exhibits a dynamic role during the action potential. Although speculative, low NKA activity during diastole would lead to accumulation of Na+. This would prime the NCX for reverse mode exchange (Ca2+ entry) during the initial phase of the action potential. This role was recently predicted in a mathematical model of excitation–contraction coupling.2 Furthermore, both the steep voltage dependence and the high affinity of the {alpha}2-isoform to Na+ reported by Horisberger and Kharoubi-Hess31 would rapidly activate the NKA during the action potential, favouring forward mode operation of the NCX to extrude Ca2+. Further evidence is needed to confirm such a dynamic role of the NKA.

In our experiments (Figure 7), NCX was forced into reverse mode, thus switching between the two exchange modes could not be studied. However, the reduced influence of NKA activation on INCX after downregulation (CHF) or inhibition (ouabain) of the {alpha}2-isoform clearly demonstrates that cross-talk between NKA and NCX was attenuated during heart failure. A higher [Na+]ss in CHF near NCX due to {alpha}2-isoform downregulation would favour NCX reverse mode exchange, and thus Ca2+ entry. This could contribute to an increased trigger of Ca2+ release, and might thus limit the contractile dysfunction due to reduced SR Ca2+ load in heart failure. It might constitute a sufficient compensatory mechanism in unloaded isolated cardiomyocytes, as indicated by the increased FS/TTP in CHF observed in this study (Figure 6B). This is supported by the observation that contraction measurements in Sham cardiomyocytes exposed to 0.3 µM ouabain converged with those in CHF cardiomyocytes. It should be noted that this might be different in the intact heart, where cardiomyocytes are exposed to an afterload. A direct comparison of the importance of {alpha}2-isoform downregulation with alterations in other Ca2+ regulatory proteins during heart failure was not possible based on the present data.

We observed that the alterations in NCX regulation observed in CHF (Figure 7B) could be mimicked in Sham by selective block of the {alpha}2-isoform. In these cells, the t-tubules were not altered. Thus, it seems likely that it is not the remodelling of t-tubules, but the loss of function of the {alpha}2-isoform, which is the main cause for altered control with NCX. However, these could act in concert in the failing heart.

This study also illustrates the importance of taking NKA activity into account when studying NCX activity. Using the same animal model, Wasserstrom et al.32 demonstrated a near doubling of reverse mode INCX in CHF. However, this could not be accounted for by the 30–40% increase in NCX protein. We propose that this discrepancy could be explained by a reduction in NKA {alpha}2-isoform, resulting in higher [Na+]ss close to NCX.

On a final note, this study has shown important consequences of downregulated NKA {alpha}2-isoform in a rat model of CHF. However, reports on the regulation of NKA {alpha}2-isoforms in different species during heart failure are divergent (for review, refer33). In particular, in human heart failure, {alpha}2-isoform expression has been reported to be unaltered, but some studies also show downregulation. Further studies are necessary to fully understand the role of the different {alpha}-isoforms of the NKA, and their regulations in the normal and failing heart.


   Funding
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 Funding
References
 
The Norwegian Council for Cardiovascular Diseases; the Norwegian Research Council, Eastern Norway Regional Health Authority; Rakel and Otto Christian Bruuns Fund; Anders Jahre’s Fund for Promotion of Science.


   Acknowledgements
 
The authors are grateful to Tævje A. Strømme for Matlab programming, Ståle Nygård for expertise with statistics, and Section for Comparative Medicine, Ullevaal University Hospital for animal care.

Conflict of interest: none declared.


   Notes
 
{dagger} This work was performed at Institute for Experimental Medical Research, Ullevaal University Hospital, Oslo, Norway. Back


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

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