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Cardiovascular Research 2001 52(3):487-499; doi:10.1016/S0008-6363(01)00412-6
© 2001 by European Society of Cardiology
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Copyright © 2001, European Society of Cardiology

Distribution and role of Na+/K+ ATPase in endocardial endothelium

Paul Fransen*, Jan Hendrickx, Dirk L Brutsaert and Stanislas U Sys

Department of Physiology and Medicine, University of Antwerp (RUCA), Groenenborgerlaan, 171, B-2020 Antwerp, Belgium

* Corresponding author. Tel.: +32-3-218-0278; fax: +32-3-218-0276 fransen{at}ruca.ua.ac.be

Received 25 May 2001; accepted 16 July 2001


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Objective: In mammalian cardiomyocytes, {alpha} isoforms of Na+/K+ ATPase have specific localisation and function, but their role in endocardial endothelium is unknown. Methods: Different {alpha} isoforms in endocardial endothelium and cardiomyocytes of rabbit were investigated by measuring contractile parameters of papillary muscles, by RT-PCR, by Western blots and by immunocytochemistry. Results: Inhibition of Na+/K+ ATPase by decreasing external K+ from 5.0 to 0.5 mmol/l caused biphasic inotropic effects. The maximal negative inotropic effect at external K+ of 2.5 mmol/l was significantly larger in +EE muscles (with intact endocardial endothelium) than in -EE muscles (with endocardial endothelium removed) (–22.5±2.4% versus –5.9±4.0%, n=7, P<0.05). Further decrease of K+ to 0.5 mmol/l caused endothelium-independent positive inotropy (27.8±11.8% for +EE versus 18.6±11.3% for –EE, n=7, P>0.05). Inhibition of Na+/K+ ATPase either by dihydro-ouabain (10–9 to 10–4 mol/l, n=4) or by K+ decrease following inhibition of Na+–H+ exchanger by dimethyl-amiloride (50 µmol/l, n=6) caused endothelium-independent positive inotropic effects only. RT-PCR and Western Blot demonstrated {alpha}1 and {alpha}2 Na-K-ATPase isoforms in cardiomyocytes, but only {alpha}1 in cultured endocardial endothelial cells. Immunohistochemistry showed that {alpha}1 in endocardial endothelium was predominantly present at the luminal side of the cell (n=7) and that {alpha}1 and {alpha}2 displayed different localisation in cardiomyocytes. Conclusions: These results suggested that negative and positive inotropic effects of Na+/K+ ATPase inhibition in +EE muscles could be attributed to inhibition of endocardial endothelial {alpha}1 and muscle {alpha}2 isoform, respectively. Accordingly, the endocardial endothelial {alpha}1 isoform of Na+/K+ ATPase may contribute to blood–heart barrier properties of this endothelium and may control cardiac performance via endothelial Na+/H+ exchange.

KEYWORDS PECAM: platelet endothelial cell adhesion molecule; P: polyclonal antibody; M: monoclonal antibody; NCE: Na+/Ca2+ exchanger; NHE: Na+/H+ exchanger; UB: Upstate Biotechnology; ABR: Affinity Biotechnology Reagents


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
Cardiac endothelial cells modulate cardiac performance by release of endothelium-derived inotropic substances [1–3]. Endocardial endothelial cells, which line the entire luminal surface of the cardiac cavities, may permit active solute transport between lumen and subendothelium. The asymmetrical distribution of ion channels between luminal and abluminal membranes of endocardial endothelial cells [4,5] suggests transendothelial ionic gradients, which may control ionic homeostasis of the subendocardial interstitium, a prerequisite for normal activity of the heart. An asymmetrically localised sodium–potassium ATPase (Na+/K+ ATPase) would contribute to these transendothelial ionic gradients [3].

Na+/K+ ATPase is a membrane-bound enzyme that consists of two subunits: a large ouabain-sensitive polypeptide ({alpha}) and a smaller glycoprotein (β). In mammalian cardiac muscle, {alpha}1, {alpha}2 and {alpha}3 are the most prominent {alpha}-isoforms with different localisation and function within a single cardiomyocyte [7,8]. They have different sensitivity to inhibition by ouabain, external pH and internal Ca2+, to activation by internal Na+ and external K+ [6,9], to {alpha}- and β-adrenergic agonists [10] and to aldosterone [11].

Electrophysiological measurements supported the existence of Na+/K+ ATPase in cardiac endothelial cells [12–14], but isoform type, localisation and function had not been studied. Endothelial cells from mouse brain and human umbilical vein expressed {alpha}1 isoforms (75–80%) [15,16] mainly. In epithelial cells {alpha}1 isoform acts as the housekeeping Na+/K+ ATPase, regulating bulk transport of Na+ and K+, and, thereby, contributing to apical-basal polarity, which has been demonstrated in endothelial cells as well, especially at the blood–brain barrier and in corneal endothelial cells [17–21]. As a result, this endothelial monolayer regulates unidirectional flux of ions between blood and cerebrospinal fluid in the brain, thereby controlling the ionic environment and electrical activity of neurones.

Here, we report the presence of an asymmetrically distributed {alpha}1 Na+/K+ ATPase subunit isoform at the luminal membrane of rat and rabbit endocardial endothelium. This suggests an important role of the polarised endocardial endothelial sodium pump in the control of the ionic environment and performance of subjacent cardiomyocytes, terminal Purkinje fibre network and subendocardial neural plexus.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Mechanical performance
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). Isometric and isotonic contractions were measured in papillary muscles isolated from the right ventricle of rabbits. The muscles were mounted vertically in an organ bath filled with modified Krebs–Ringer solution (mKR) containing in mmol/l: 98 NaCl, 4.7 KCl, 2.4 MgSO4.7H2O, 17 NaHCO3, 1.2 KH2PO4, 4.5 glucose, 1.8 CaCl2, 5 CH3COONa.3H2O, 15 C3H3O3Na, 0.02 atenolol, 3 2,3-butanedionemonoxime (C4H7NO2, BDM), 5% bovine serum and gassed with a mixture of 95% O2–5% CO2 at 35.0±0.5°C and pH of 7.4±0.1. All experiments were performed in two groups of muscles. In one group, the endocardial endothelium was selectively destroyed (–EE) immediately following dissection of the muscles by immersion for 1 second in a 0.5% Triton X100 mKR, followed by abundant wash with mKR. Subsequently, these muscles were treated in the same way as the muscles from the group with intact endocardial endothelium (+EE). Immunostaining of the muscles with an antibody for platelet endothelial cell adhesion molecule (PECAM, monoclonal mouse anti-human CD31, DAKO, Denmark) revealed that +EE muscles were lined by an intact endocardial endothelium, while the surface of –EE muscles stained negatively for PECAM.

After 10 min, muscles were stimulated at interstimulus interval of 1670 ms and voltage of 10% above treshold by rectangular pulses of 5 ms duration through two platinum electrodes. Then, BDM was removed and the muscle started to contract. Muscles were allowed to stabilise for 20 min, after which lmax (the muscle length at which active force development was maximal) was determined and the solution was switched to a normal serum-free KR with the same composition except that NaHCO3 was changed from 17 mmol/l to 20 mmol/l to keep the pH at 7.4±0.1. Changes of external K+ ([K+]0) were achieved by mixing a zero K+ KR, in which 5.9 mmol/l K+ was replaced by 5.9 mmol/l Na+, and the standard KR. Dihydro-ouabain (DHO) and dimethylamiloride (DMA) were prepared from freshly made stock solutions. The following contractile parameters from an isotonic preloaded and a fully isometric twitch were determined: peak isotonic twitch shortening, resting tension, total peak isometric twitch tension, peak rate of change of twitch tension ((dF/dt)max) and time from stimulus to half-isometric twitch relaxation (tHR). At each experimental intervention, isotonic and isometric twitches were measured at the basal stimulation frequency (36 contractions/min) and following paired stimulation, in which the paired stimulus was moved closer to the basal stimulus at frequency of 36 contractions/min until shortening or isometric force of the basal beat was maximal. Force measurements were normalised for muscle cross-sectional area, which was calculated at the end of the experiment by dividing the lightly blotted wet weight of the muscle by its length at lmax (a cylindrical shape and a specific gravity of 1.0 were assumed). Basal characteristics for +EE muscles (n=7) were 4.6±0.3 mm for lmax, 0.88±0.18 mm2 for cross sectional area, 8.3±1.6 mN/mm2 for preload, 24.9±7.6 mN/mm2 for total peak isometric tension, 315±20 ms for tHR and 190±52 mN/mm2/s for (dF/dt)max. These values were not significantly different (P>0.5) for –EE muscles (n=7).

All data are expressed as mean±S.E.M. Repeated measures analyses of variance (SPSS 9.0, SPSS, Chicago) were performed on the raw data (after logarithmic transformation for homoscedasticity) in order to analyse the effects of K+, and the influences of an intact endocardial endothelium, DMA and the mode of electrical stimulation. Differences were considered statistically significant when P<0.05.

2.2 Isolation of rabbit cardiac myocytes
Single rabbit myocytes were isolated by an enzymatic isolation procedure [22]. Hearts were retrogradely perfused through the aorta with the following solutions: 1: normal KR but 1 mmol/l MgCl2 and 5 mmol/l HEPES for 5 min; 2: Ca2+-free KR (see above, but 0.5 mmol/l MgCl2 and 5 mmol/l HEPES) for 10 min; 3: Ca2+-free KR containing 50 mg/100 ml collagenase A (Boehringer) and 25 mg/100 ml trypsin inhibitor (Boehringer) for 8–9 min; 4: Ca2+-free KR of 3 with 4 mg/100 ml protease type XIV (Sigma) for 3–4 min; 5: Ca2+-free storage buffer solution with (in mmol/l): 40 KCl, 20 KH2PO4, 50 L-glutamic acid, 20 taurine, 0.5 EGTA and 10 glucose, pH 7.2 (KOH) for 5 min. All solutions (except 5) were gassed with 5% CO2–95% O2). Subsequently, the heart was removed from the perfusion apparatus; the ventricles were cut in small pieces and gently agitated in the storage buffer solution, which was then sieved and centrifuged to isolate single myocytes.

2.3 RT-PCR
RNA was extracted from cultured rabbit endocardial endothelial cells by adding 2 ml of TriZol reagent (Life Technologies) directly to the 20 cm2 culture disk, from centrifuged rabbit isolated ventricular myocytes by adding 2 ml of TtiZol reagent directly to the centrifuged cell suspension and from rabbit heart and brain tissue by disrupting 50 to 100 mg tissue in 1 ml of TriZol with a polytron rotor homogenizer. Subsequently, the RNA was isolated according to the manufacturer’s instructions. Concentrations were determined by measuring the absorbency at 260 nm.

RT-PCR was performed using the One-Step RT-PCR System (Life Technologies) in a 25 µl reaction volume containing 0.5 µl total RNA (10–100 ng) and 400 nmol/l of primers for the {alpha}1, {alpha}2 and {alpha}3 isoforms of the Na+/K+ ATPase and for isoform 1 of the Na+/H+ exchanger (NHE, Table 1) according to the manufacturer’s instructions. After an initial incubation for 30 min at 50°C and 2 min at 94°C, 35 cycles of PCR were performed consisting of a 15 s denaturation at 94°C, a 30 s primer annealing at 50 to 55°C, and a 1 min extension at 72°C, with a final extension step of 8 min. The PCR products were analyzed on a 1.8% LSI agarose gel (Life Sciences International).


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  		Table 1 PCR Primer sets for {alpha} isoforms of Na+/K+ ATPase and for Na+/H+ exchanger (isoform 1, NHE1)

 
2.4 Western blot
Heart (cardiac muscle tissue or endocardial endothelial cell culture) proteins (20–80 µg for immunological detection of {alpha}1 and {alpha}2 isoforms) and brain and/or kidney proteins (40 µg rat brain or kidney microsomal protein preparation, Upstate Biotechnology, USA) were loaded on a 7.5 or 12% Tris–HCl gel. Following electrophoretic separation, proteins were transferred to a nitrocellulose-membrane (Amersham, UK) at 100 V for 1 h. Protein containing membranes were incubated with blocking solution (5% fat-free milk, 0.1% Tween-20 in Tris-buffered saline) for 1 h at room temperature. Specific primary antibodies ({alpha}1 fusion protein rabbit polyclonal IgG, anti rat Na+/K+ ATPase Kit 2, Upstate Biotechnology, USA, and mouse monoclonal anti {alpha}2 Na+/K+ ATPase, a kind gift by Dr. K. Sweadner, USA) were diluted in blocking solution plus thimerosal (1:1000 for {alpha}1 and {alpha}2) and applied overnight at room temperature. Incubation with secondary antibodies, horseradish peroxidase-conjugated goat anti-rabbit IgG antibodies (diluted 1:10,000) was performed for 1 h at room temperature. The reactive proteins were detected by chemiluminescent reaction followed by exposition of the membranes to Hyperfilm (Amersham, UK).

2.5 Immunocytochemistry
Rat hearts or rabbit left or right ventricles were kept overnight in a saturated sucrose solution and frozen (Tissue-Tek) for cryostat sections, which were air-dried for 20 min and, then, fixated with cold acetone (10 min, 4°C). The cryostat sections were rinsed with phosphate buffered saline (PBS, 5 min), put in glycine (10 min), washed in PBS, and placed in blocking solution (30 min, 50 µl goat IgG/10 ml bovine serum albumine solution) and finally in washing buffer (5 min), all at room temperature. Following incubation overnight at 4°C with the primary antibody (Table 2) and 3 times wash (10 min) with PBS, they were incubated for 2 h at 37°C with secundary antibody (goat anti-mouse or goat anti-rabbit FITC or cy3, Jackson ImmunoResearch Lab, USA). The cryostat sections were placed again in washing buffer (15 min), washed 3 times (5 min) in PBS and mounted in Slowfade plus glycerol (Molecular Probes, USA). Images were made at 100x magnification on an Olympus (U-RFL-T) or Reichert Polyvar II (Leica) epifluorescence microscope with a Sony 3 CCD (DXC-9100P) or SensiCam 12 bit cooled CCD (PCO, Germany) and stored on computer for later analysis with Analysis or Photoshop software.


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  		Table 2 Primary antibodies for immunocytochemistry

 

   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 Inotropic effects of external K+ reduction
The functional demonstration of Na+/K+ ATPase in endocardial endothelium and in ventricular myocardium was assessed in isolated rabbit papillary muscles. At external K+ concentrations ([K+]0) from 5 down to 0.5 mmol/l to inhibit Na+/K+ ATPase [9], the inotropic parameters, (dF/dt)max and tHR were determined in –EE and +EE muscles (n=7). Decrease of [K+]0 in –EE and +EE muscles caused biphasic effects on (dF/dt)max and tHR (Figs. 1 and 2Go). In –EE muscles, a decrease in [K+]0 from 5 down to 2.5 mmol/l, caused a small but non-significant negative inotropic effect (–5.9±4.0%, P>0.05). The negative inotropic effect of lowering [K+]0 in +EE muscles, however, reached –22.5±2.4% at 2.5 mmol/l [K+]0 (P<0.05) and was significantly more pronounced than in –EE muscles (P=0.025). Further decrease in [K+]0 increased (dF/dt)max by 18.6±11.3% in –EE and to 27.8±11.8% in +EE muscles at 0.5 mmol/l [K+]0 (Fig. 2A). The latter values were similar for +EE and –EE groups. Resting tension in all muscles (n=13) increased by 41.3±5.6% (n=7, P<0.001) at [K+]0 of 0.5 mmol/l (data not shown). For tHR (Fig. 2C), the effect of [K+]0 decrease was also endothelium-dependent (P<0.0005), with tHR being significantly shorter in +EE muscles between 3 and 1.5 mmol/l [K+]0 than at 5 mmol/l [K+]0 (P<0.05).


Figure 1
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  		Fig. 1 Original data of representative isometric twitches at 5.0, 2.5 and 1.0 mmol/l [K+]0 are shown in A through D in two muscles (–EE: endocardial endothelium removed and +EE: intact endocardial endothelium) in the absence (A,C) and presence (B,D) of 50 µmol/l dimethylamiloride (DMA). The stimulation frequency was 36/min.

 

Figure 2
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  		Fig. 2 Mean peak rate of isometric twitch force development ((dF/dt)max, A,B) and time from stimulus to half isometric twitch relaxation (tHR, C,D) as a function of [K+]0 in –EE muscles (closed symbols) and +EE muscles (open symbols) in control conditions (circles, A,C, n=7) and following addition of 50 µmol/l DMA (squares, B,D, n=6). (dF/dt)max and tHR are expressed as percentage of baseline value at 5 mmol/l [K+]0 in control (absence of DMA) conditions in –EE and +EE muscles, which was taken as 100%. Note the different scaling of the y-axis. Asterisks indicate significant difference from the baseline value at 5 mmol/l external K+ (P<0.05). P-values for statistical comparison of [K+]0 curves between +EE and –EE muscles are indicated between the curves.

 
Negative inotropy has often been associated with changes in pH [23]. Therefore, the possible contribution of the Na+/H+ exchanger (NHE) to the negative inotropic effects caused by inhibition of Na+/K+ ATPase at concentrations below 5 mmol/l [K+]0 was investigated by adding 50 µmol/l dimethylamiloride (DMA, a selective inhibitor of NHE) prior to decreasing [K+]0 (Figs. 1BGo,D and 2B,D). Inhibition of NHE by DMA at basal [K+]0 of 5 mmol/l, did not affect (dF/dt)max in +EE muscles (–1.1±7.1%) and slightly decreased (dF/dt)max in -EE muscles (–10.1±10.5%) (n=6). When [K+]0 was decreased in the presence of DMA, only positive inotropy was observed in both +EE and –EE muscles; this effect was endothelium-independent (P=0.996 for dF/dtmax and P=0.86 for tHR) (Figs. 2B,D). The increase in resting tension, observed in the absence of DMA at the lowest [K+]0 of 0.5 mmol/l, was significantly inhibited by DMA to +10.1±4.9% (data not shown, P=0.005).

3.2 Inotropic effects of dihydro-ouabain (DHO)
Specific inhibition of Na+/K+ ATPase by dihydro-ouabain (DHO) caused positive inotropic effects only, regardless of whether experiments were performed at [K+]0 of 5 mmol/l (+EE muscles) or of 2 mmol/l (+EE and –EE muscles). To obtain dose–response curves of DHO, (dF/dt)max-values were expressed relative to (dF/dt)max-values measured during paired stimulation (as an estimate of the maximal inotropic response). The apparent Kd value of DHO for the positive inotropic effect (32.5±13.2 µmol/l, n=4) at 5 mmol/l [K+]0 in +EE muscles was not significantly different from apparent Kd values at 2 mmol/l [K+]0 in +EE muscles (184±121 µmol/l, n=5) and –EE muscles (50.4±7.2 µmol/l, n=3) (P>0.05). DHO (10–4 mol/l) increased resting tension by 23.6±3.2% (n=6, data not shown). The positive inotropic effects of DHO were endothelium- and [K+]0-independent (P>0.05).

Accordingly, results in rabbit papillary muscles suggested that different isoforms of Na+/K+ ATPase might be present in endocardial endothelial cells and in cardiomyocytes and that endocardial endothelial cells displayed NHE as well. We further investigated Na+/K+ ATPase isoforms and NHE at the mRNA and protein level.

3.3 Presence of {alpha}1 and {alpha}2 mRNA and protein in rabbit cardiac tissue
RT-PCR, which was performed on RNA of rabbit cultured endocardial endothelial cells, freshly isolated rabbit ventricular cardiomyocytes and fresh myocardial tissue with {alpha}1, {alpha}2, {alpha}3 and NHE primer sets, clearly showed the presence of {alpha}1 and {alpha}2 subunit isoforms in isolated rabbit cardiomyocytes as well as the presence of the {alpha}1 subunit in cultured endocardial endothelial cells. However, {alpha}2 was absent in cultured endocardial endothelium (Fig. 3A). The {alpha}3 subunit mRNA (data not shown) was present in rabbit brain, but not in total heart tissue or in endocardial endothelium. NHE mRNA was present in rabbit total heart tissue as well as in cultured endocardial endothelial cells (Fig. 3B).


Figure 3
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  		Fig. 3 (A) RT-PCR of {alpha}1 and {alpha}2 subunit isoforms of Na+/K+ ATPase in isolated rabbit cardiomyocytes (C) and rabbit cultured endocardial endothelial cells (E). M=molecular weight marker. Marker fragment lengths are shown on the left. The {alpha}1 and {alpha}2 primers amplify a 289 bp and 205 bp fragment of rabbit {alpha}1 and {alpha}2 subunits, respectively. (B) RT-PCR of Na+/H+ exchanger (NHE1) isoform in rabbit cardiac muscle (H) and rabbit cultured endocardial endothelial cells (E). The NHE1 primers amplify a 297 bp fragment of rabbit NHE1. M=molecular weight marker. Marker fragment lengths are shown on the left. Cardiac muscle (H) refers to intact cardiac tissue consisting of various cell types, including mostly cardiomyocytes, but also vascular endothelial cells, fibroblasts etc.

 
Western blot analysis of Na+/K+ ATPase {alpha}1 and {alpha}2 isoforms (Fig. 4) revealed the presence of the {alpha}1 isoform in cultured endocardial endothelium of rabbit right ventricle as well as in fresh rabbit myocardium, whereas the {alpha}2 isoform was only detected in myocardial preparations (n=3).


Figure 4
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  		Fig. 4 Western Blot for {alpha}1 and {alpha}2 subunit isoforms of Na+/K+ ATPase in rabbit cultured endocardial endothelial cells (E) and rabbit isolated cardiac muscle (H). Molecular weight marker is shown on the right, while the amount of applied protein is indicated under the blots. As controls, 40 µg of brain protein (for {alpha}1) and of brain and kidney protein (for {alpha}2) were applied.

 
3.4 Localisation of Na+/K+ ATPase isoforms in endocardium
Previous results suggested that, in muscle cells, there was a link between the {alpha}2 subunit isoform of Na+/K+ ATPase and the Na+/Ca2+ exchanger (NCE, positive inotropy) [8,24] and in endocardial endothelium between the {alpha}1 subunit isoform of Na+/K+ ATPase and NHE (negative inotropy). Therefore, localisation of Na+/K+ ATPase {alpha}1 and {alpha}2 isoforms and of NHE and NCE was investigated in transverse cryostat sections through whole rat hearts or rabbit left ventricles by immunofluorescent staining with polyclonal and monoclonal antibodies (Table 2).

Immunostaining of rat ventricle for PECAM (platelet endothelial cell adhesion molecule) and {alpha}1 Na+/K+ ATPase revealed that the {alpha}1 isozyme was mainly present at the luminal side of endothelial cells (n=7, Fig. 5). No evidence for the presence of {alpha}2 (Fig. 6F) or of NCE (Fig. 6A) in endocardial endothelium was found. The exact localisation of NHE (luminal or abluminal) could not be determined in these experiments (Fig. 6D,F). Staining of cardiomyocytes with the {alpha}1 Na+/K+ ATPase isoform antibody occurred mainly at cell borders and at intermyocytal blood vessels (Fig. 6B,D,E), where there was some co-staining with NCE (Fig. 6B) and NHE (Fig. 6E). The {alpha}2 Na+/K+ ATPase isoform was clearly evident at the T-tubules (Fig. 6C,G), suggesting a different localisation and function for {alpha}1 and {alpha}2 isoforms in rat cardiomyocytes. The NCE was present along the entire plasmalemma (Fig. 6B,C) and at the T-tubules (Fig. 6A). The NHE was present mainly along cell borders, intermyocytal blood vessels and nuclei of cardiomyocytes (Fig. 6D–G).


Figure 5
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  		Fig. 5 Double immunostaining of cryostat sections of rat heart for PECAM (platelet endothelial cell adhesion molecule, A, red) and {alpha}1 isoform of the Na+/K+ ATPase (B, green). PECAM outlined endocardial endothelial cells especially at sites of cellular overlap and also stained the abluminal side of cells as seen at the nuclei (arrows, asterisks). {alpha}1 isoform staining was present at the luminal side of endocardial endothelial cells only, which was evident at the site of the cell nuclei (green arrows). C combines figures A and B; overlap between {alpha}1 isoform and PECAM staining is indicated by yellow, which is absent at the abluminal side of endocardial endothelial cells.

 

Figure 6
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  		Fig. 6 Double immunostaining of cryostat sections of rat heart for {alpha}1 or {alpha}2 isoforms of Na+/K+ ATPase (green) and NCE or NHE (red). The respective combinations are indicated in the figures. Overlap between green and red staining is indicated by yellow. L indicates ventricular lumen. The {alpha}1 isoform was present at the luminal side of the endocardial endothelium, where there was no staining for {alpha}2 (G,I) or NCE (A). In cardiomyocytes, staining for {alpha}1 isoform (B,E) and NCE (B,C) was present at the T-tubules and along the cell borders (especially {alpha}1), where there was some overlap with NCE (B). Overlap between {alpha}1 isoform and NCE staining occurred at the site of the blood vessels also (black arrows, B). The {alpha}2 isoform was present at the T-tubules only (C,G-I). NHE was present in the luminal and abluminal membrane of endocardial endothelial cells (G). In cardiomyocytes, NHE was present mainly at the cell borders (D–I), but also at the T-tubules (I). Overlap with {alpha}1 isoform staining was evident at the cell borders (E–F) and blood vessels (black arrow in E), whereas some overlap with {alpha}2 isoform staining was observed also at the T-tubules (I).

 
In rabbit, cryostat sections of left ventricle were stained for {alpha}1, {alpha}2, NCE, NHE and triadin (Fig. 7). Triadin is an integral membrane protein of the sarcoplasmic reticulum, shown to interact with the ryanodine receptor, junctin and calsequestrin [25]. Staining for triadin helps to compare localisation of Na+/K+ ATPase, NCE and NHE with respect to the T-tubules (triadin) in myocardium. In endocardial endothelium, only {alpha}1 and NHE were observed. The {alpha}1 isoform was mainly present at the luminal side of the cell (as in rat). The exact localisation of NHE in rabbit could not be determined, although the absence of positively stained nuclear extrusions might suggest abluminal localisation. In muscle, staining of mid-myocardium with the different monoclonal antibodies revealed again that {alpha}1 and {alpha}2 isoforms displayed different localisation. Positive staining for {alpha}1 was present mainly along cell borders (as in rat), where there was also strong staining for NCE (stronger than rat) and NHE. Triadin and {alpha}2 staining displayed similar patterns, indicating the almost exclusive presence of {alpha}2 at the junctions of plasmalemma with the sarcoplasmic reticulum (triadin staining pattern). NCE and NHE were present also at the junctional membranes of plasmalemma and sarcoplasmic reticulum.


Figure 7
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  		Fig. 7 Immunostaining of cryostat sections through left ventricle of rabbit heart with left hand Figs. showing endocardial images, and right hand figures showing mid-myocardial images. Sections were stained for {alpha}1 and {alpha}2 isoforms of Na+/K+ ATPase, NCE, NHE and triadin (T). L indicates ventricular lumen. In endocardial endothelial cells, {alpha}1 (luminal localisation) and NHE (abluminal localisation) were present only (arrows, A,G). In cardiomyocytes, {alpha}1(B), NCE (F) and NHE (H) were present along the cell borders, whereas {alpha}2 (D) and T (J) were only evident as spots, indicating the junctional complexes of sarcolemma and sarcoplasmic reticulum. Finally, NCE (E,F) and NHE (G,H) were present also at the junctional complexes.

 

   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
The present study demonstrated that inhibition of endocardial endothelial Na+/K+ ATPase by reducing [K+]0 caused negative inotropy. The effect was attenuated by inhibition of the endothelial NHE, suggesting coupling between both ion transport systems. By contrast, inhibition of cardiac muscle Na+/K+ ATPase by reducing [K+]0 or by adding dihydro-ouabain caused positive inotropy, probably via the muscle NCE.

4.1 Different localisation of {alpha}1 and {alpha}2 Na+/K+ ATPase isoforms
Rabbit endocardial endothelial cells expressed and displayed the {alpha}1 isoform of Na+/K+ ATPase, but not {alpha}2 or {alpha}3 isoforms. On immunohistochemistry this {alpha}1 isoform co-localised with PECAM (in rat) and was asymmetrically distributed between luminal and antiluminal side of the cell. Similarly [26] or contrary [19] to its localisation in blood–brain barrier endothelial cells, the {alpha}1 isoform was mainly present at the luminal side of the endocardial endothelial cell. This suggests a role of endocardial endothelium as a blood–heart barrier controlling the ionic environment of subjacent myocytes. Similarly to guinea pig [10,27], adult rat [28] and mouse [8], cardiomyocytes of rabbit expressed and displayed {alpha}1 as well as {alpha}2 (but not {alpha}3) Na+/K+ ATPase isoforms (see, however, [29]). In rabbit as well as in rat ventricle, {alpha}1 and {alpha}2 isoforms displayed different localisation in cardiomyocytes. The {alpha}1 isoform was homogeneously distributed along the plasmalemma of cardiomyocytes. Co-localisation with plasmalemmal NCE and NHE suggests regulation of bulk transport of Na+, K+, H+ and Ca2+ and restoration of basal intracellular K+, Na+ and Ca2+ following action potentials [6,8,24]. The {alpha}2 isoform was not present over the entire plasmalemma of cardiomyocytes, but was clearly evident along the T-tubules. The isoform co-localised with triadin and with the NCE and NHE, which suggested a role for the {alpha}2 isoform of the Na+/K+ ATPase in local Ca2+ signalling and contractility [6,8,24].

4.2 Inhibition of {alpha}1 and {alpha}2 Na+/K+ ATPase isoforms
In our study, inhibition of Na+/K+ ATPase by reduction of [K+]0 revealed biphasic inotropic effects: endothelium-dependent negative inotropic effects with a maximum around 2.5 mmol/l [K+]0 and endothelium-independent positive inotropic effects below 2 mmol/l [K+]0. Biphasic effects by inhibition of different Na+/K+ ATPase isoforms were observed also in mouse hearts with genetically reduced levels of Na+/K+ ATPase isoforms (heterozygous {alpha}1+/– or {alpha}2+/– hearts) [8]. Heterozygous {alpha}1+/– hearts and {alpha}2+/– hearts were hypo- and hypercontractile respectively. Hypercontractility was due to increased Ca2+ transients in cardiomyocytes isolated from {alpha}2+/– hearts. Local Na+ increase by {alpha}2 inhibition elicits Ca2+ influx via NCE [6,8,24]. In single cardiomyocytes isolated from hypocontractile {alpha}1+/– hearts (hence in the absence of endothelial cells) no change in intracellular Ca2+ transients was observed. This was similar to our results in –EE muscles, where the negative inotropic effect of [K+]0 reduction on (dF/dt)max, which may be a measure of internal Ca2+ availability [30,31], was only 5%. In +EE muscles, however, the negative inotropic effect on (dF/dt)max was about –20%, similar to the value in heterozygous {alpha}1+/– hearts. It is, therefore, suggested that for depressed contractility in {alpha}1+/– hearts or in rabbit papillary muscles to become manifest, an intact endothelium with {alpha}1 as main Na+/K+ ATPase isoform is obligatory.

That the negative inotropic effect of a moderate decrease in [K+]0 in +EE muscles is due to endocardial endothelial pump inhibition was based on the following observations. (1) With molecular techniques, it was shown that endocardial endothelial cells displayed the {alpha}1 isoform only. (2) The negative inotropic effect at 3 to 2 mmol/l [K+]0 was endothelium-dependent and occurred in the [K+]0 range where {alpha}1 is blocked. In guinea-pig cardiomyocytes, [K+]0 activated {alpha}1 isoform of Na+/K+ ATPase half-maximally at 3.7 mmol/l [K+]0 [9]. This concentration fits within the concentration range of [K+]0 of 5.0–2.0 mmol/l, which in the present study gave rise to negative inotropy in rabbit papillary muscles and also with the concentration of 2.7 mmol/l [K+]0 at which it had been shown previously with electrophysiological techniques that inhibition of an active Na+/K+ ATPase in endocardial endothelial cells had occurred [14].

Several observations suggested that the positive inotropic effect of a more pronounced decrease in [K+]0 in +EE and –EE muscles was due to Na+/K+ ATPase inhibition in cardiomyocytes. (1) With molecular techniques, it was shown that rabbit cardiomyocytes, not endocardial endothelial cells, displayed the {alpha}2 isoform. (2) The positive inotropic effect at [K+]0 below 2 mmol/l was endothelium-independent and occurred in the [K+]0 range where {alpha}2 is blocked [9]. (3) The effects of low [K+]0 were similar to the effects of dihydro-ouabain (DHO), i.e. with endothelium-independent positive inotropy, twitch prolongation and increase in resting tension. In guinea-pig, canine and rat cardiomyocytes, the Kd-value of ouabain is less than 1 µmol/l for the {alpha}2 isoform and exceeds 50 µmol/l for the {alpha}1 isoform [6,9]. The differences in cardiotonic steroid affinity may not be as marked in rabbit [6]. It is, therefore, expected that DHO at concentrations below 100 µmol/l inhibited mainly the {alpha}2 isoform. The apparent Kd value for ouabain of about 30 µmol/l in rabbit was 40 to 50 times higher than the apparent Kd value of 0.75 µmol/l in guinea pig and canine cardiomyocytes [9].

4.3 The endocardial endothelial {alpha}1 isoform and NHE
The negative inotropic effect of endocardial endothelial {alpha}1 inhibition on (dF/dt)max and on tHR in +EE muscles was suppressed in the presence of DMA and was, thus, mediated via activity of NHE. Furthermore, the effects of [K+]0 decrease in the presence of DMA had become endothelium-independent suggesting that the endothelial NHE was involved. Accordingly, the endocardial endothelial {alpha}1 isoform of the Na+/K+ ATPase via the endothelial NHE controls cardiac performance and, thus, internal Ca2+ of the subjacent cardiomyocytes. It is hypothesised, therefore, that the endothelial NHE might be responsible for a lower intermyocytal pH. This might be compatible with the observations of an endocardial-to-subendocardial interstitial and intracellular pH gradient in perfused rabbit papillary muscles [32]. It would be interesting to know if the endothelial NHE and/or Na+/K+ ATPase contribute to this gradient. The present study in rat and rabbit showed the presence of NHE in the endocardial endothelium (probably at the abluminal side as observed in human pulmonary artery endothelial cells [33]), in the plasmalemma and T-tubules of cardiomyocytes and in the myocardial capillaries. Co-localisation with NCE suggests effects on Ca2+ mobilisation and excitation-contraction coupling [34,35].

In conclusion, molecular and morphological techniques confirmed the different distribution of {alpha}1 and {alpha}2 isoforms of Na+/K+ ATPase between endocardial endothelium and cardiomyocytes. Endocardial endothelial cells expressed {alpha}1 isoforms only, which is the ubiquitous Na+/K+ ATPase isoform in endothelial cells [15,16], suggesting transport function. The {alpha}1 isoforms were present at the luminal side of the cell, leading to transendothelial transport of Na+ and K+. As a consequence, intermyocytal K+ is higher and intermyocytal Na+ is lower than their respective plasma concentrations (Fig. 8). The asymmetrical distribution of ion channels [4,5], the abluminal NHE and Na+/HCO3– cotransporter [36,37] and the luminal {alpha}1 Na+/K+ ATPase may contribute to barrier properties of the endocardial endothelium providing further evidence for the concept that the endocardial endothelium functions as an active blood–heart barrier, which controls subendothelial and global myocytal concentration of Na+, K+ and H+. The sensitivity of this barrier to plasma K+ could rise still another hypothesis, namely that the endocardial endothelium would act as some kind of a sensor device for various ions in the plasma. Inhibition of endocardial endothelial {alpha}1 Na+/K+ ATPase leads to attenuated transendothelial K+ and Na+ transport, an increase in intermyocytal Na+ and H+ and a decrease in intermyocytal K+. External acidification via stimulated NHE may explain the observed negative inotropy. How NHE is stimulated at low external K+ or following endocardial endothelial {alpha}1 isoform inhibition needs further investigation. Endothelial acidification via attenuated Na+/HCO3– cotransport at low external K+ might be involved [38]. In cardiomyocytes, the different localisation of {alpha}1 at the plasmalemma and {alpha}2 at the T-tubular membrane suggests different functions of the isoforms with {alpha}1 regulating global Na+, K+ and Ca2+ restoration following an action potential and {alpha}2 regulating local Na+, K+ and Ca2+ concentrations [7,8,23]. Inhibition of myocytal {alpha}1 Na+/K+ ATPase causes only minor negative inotropic effects, whereas inhibition of myocytal {alpha}2 Na+/K+ ATPase leads to an increased myocytal Na+ and via NCE to increased myocytal Ca2+ and positive inotropy. As cardiac Na+/K+ ATPase has been ascribed a key role in a number of pathophysiological conditions such as hypoxia–reoxygenation, ischemia–reperfusion, primary hypertension, hypertrophy and heart failure [24,26,39], one may wonder from the present results whether and to what extent endocardial endothelial Na+/K+ ATPases are involved.


Figure 8
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  		Fig. 8 Endocardial endothelial modulation of subendocardial myocardial performance via Na+/K+ ATPase. The {alpha}1 subunit isoform of Na+/K+ ATPase in endocardial endothelial cells is located at the luminal side of the cells. Together with the abluminal NHE and Na+/HCO3– cotransporter [37,38] and luminal or abluminal ion channels, subendothelial and global myocytal concentration of Na+, K+ and H+ are controlled. Inhibition of endocardial endothelial {alpha}1 Na+/K+ ATPase leads to increased intermyocytal H+ via stimulated NHE, which may explain the observed negative inotropy. In cardiomyocytes, the different localisation of {alpha}1 at the plasmalemma and {alpha}2 at the T-tubular membrane suggests different functions of the isoforms. Inhibition of myocytal {alpha}1 Na+/K+ ATPase causes only minor negative inotropic effects, whereas inhibition of myocytal {alpha}2 Na+/K+ ATPase leads to an increased myocytal Na+ and via NCE to increased myocytal Ca2+ and positive inotropy. (IRK+: inwardly rectifying K+ channel; K+Ca: Ca2+-dependent K+ channel; cation: non-selective cation channel; Cl–: outwardly rectifying background Cl- current).

 
Time for primary review 21 days.


   Acknowledgements
 
The authors wish to acknowledge Dr. Marc Kockx (AZ Middelheim, Antwerp), Dr. Michiel Knaapen (APCAM, Antwerp), Maria Michiels and Pascale Van Tongelen for excellent technical and experimental support. This work is supported by Concerted Research Project UA 1998: ‘Endothelium and cellular infiltrate in tissue remodelling’. Jan Hendrickx is supported by Levenslijn (FWO Vlaanderen) 7.0052.98.


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

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