Cardiovascular Research

Cardiovascular Research 2007 73(1):92-100; doi:10.1016/j.cardiores.2006.11.006

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Copyright © 2006, European Society of Cardiology

Differential distribution and regulation of mouse cardiac Na⁺/K⁺-ATPase ₁ and ₂ subunits in T-tubule and surface sarcolemmal membranes

Roger G. Berry^a, Sanda Despa^b, William Fuller^a, Donald M. Bers^b and Michael J. Shattock^a,*

^aCardiac Physiology, King's College London, The Rayne Institute, St Thomas' Hospital, London SE1 7EH, UK
^bDepartment of Physiology, Loyola University Chicago, Maywood, Illinois, USA

^* Corresponding author. Tel.: +44 20 7188 0945; fax: +44 20 7188 3902. Email address: michael.shattock{at}kcl.ac.uk

Received 9 August 2006; revised 16 October 2006; accepted 7 November 2006

	Abstract

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
Objectives: Two Na⁺/K⁺-ATPase (NKA) -subunit isoforms, ₁ and ₂, are expressed in the adult mouse heart. The subcellular distribution of these isoforms in T-tubule and surface sarcolemmal (SSL) membranes and their regulation by cAMP-dependent protein kinase (PKA) is unclear.

Methods: We used formamide-induced detubulation of mouse ventricular myocytes to investigate differential functional distribution and regulation by PKA of ₁ and ₂ in T-tubule versus SSL membranes by measuring NKA current (I_pump) and NKA-mediated Na⁺ efflux (–d[Na]_i/dt).

Results: I_pump is composed of 88% ₁-mediated I_pump (I₁) and 12% ₂-mediated I_pump (I₂). ₁ and ₂ subunits demonstrate distinct ouabain affinities (105±6 and 0.3±0.1 µmol/L respectively) but similar affinity for intracellular Na⁺ (K_1/2Na⁺ of 16.6±0.8 and 16.7±2.6 mmol/L respectively). Detubulation reduced (i) I_pump density (1.42±0.1 to 1.20±0.04 pA/pF), (ii) cell capacitance (181±12 to 127±17 pF), and (iii) I₂ contribution (12 to 6%). Total I_pump density was 60% higher in T-tubule (1.94 pA/pF, derived) vs. SSL membranes. Although T-tubule membranes represent only 30% of total surface area, they generate 70% of I₂ and 37% of I₁. I₁ density was substantially higher than I₂ in SSL (I₁:I₂=16:1) but this was markedly reduced in T-tubules (4:1). In addition to differential localisation, isoprenaline (ISO, 1 µmol/L) significantly increased ₁-mediated NKA Na⁺ affinity (from 16.6±0.8 to 13.3±1.4 mmol/L) and caused a small increase in maximal NKA Na⁺ efflux rate. ISO had no effect on ₂-mediated NKA activity.

Conclusion: These data suggest that NKA ₁ and ₂ subunits are differentially localised and regulated by PKA in T-tubule and SSL membranes and may have distinct regulatory roles in cardiac excitation–contraction coupling.

KEYWORDS Ion pumps; Na/K-pump; Sarcolemmal; Protein kinase A; Adrenergic agonist

	1. Introduction

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
The cardiac Na⁺/K⁺-ATPase (NKA) is the primary mechanism by which intracellular sodium ([Na⁺]_i), and hence intracellular calcium [Ca²⁺]_i is regulated in the heart. The NKA establishes and maintains the physiological transmembrane [Na⁺] gradient which is essential for a plethora of cellular functions [17,20,26,45] and indirectly controls myocardial contractility by influencing Na⁺/Ca²⁺ exchange (NCX) activity [30,34] and indirectly setting sarcoplasmic reticulum (SR) Ca²⁺ load and contractility.

The NKA is a heteromeric enzyme composed of an subunit (112 kDa) and a glycosylated β subunit (53 kDa) [27]. The catalytic -subunit contains binding sites for Na⁺, K⁺, ATP and cardiac glycosides. Four isoforms of the -subunit have been identified and demonstrate tissue specific distribution [28,38,41,44]. It is widely reported that the existence of multiple -subunit isoforms with tissue-specific distribution, is coupled to specialised and specific physiological roles [19,31,32,40,46]. Cardiac isoform expression varies depending on species. ₁ and ₂ are expressed in rat, guinea pig and mouse heart [15,22], while three isoforms (₁, ₂, and ₃) are present in human heart [23,39]. Experimentally, NKA ₁ and ₂ activity can be distinguished based on their differing sensitivity to cardiac glycosides. In voltage-clamped guinea-pig ventricular myocytes, Gao et al. [14] demonstrated a clear biphasic relationship between increasing concentration of dihydro-ouabain (DHO) and inhibition of whole-cell Na⁺/K⁺ pump current (I_pump). This biphasic relationship was due to the presence of both high DHO affinity ₂ pumps and low affinity ₁ pumps.

In physiological terms, it has been proposed that the Na⁺/K⁺-ATPase may be specifically tailored for a tissue by differential expression of a mix of functionally different pump isoforms [14]. Studies by Lingrel and colleagues investigated the possibility that -subunit isoforms are functionally and spatially distinct in the mouse heart. Measurement of cardiac contractility in Langendorff perfused mouse hearts with genetically reduced levels (50%) of cardiac Na⁺/K⁺-ATPase ₁ or ₂ isoforms lead to the proposal of a compartmentalisation model whereby ₂ regulates [Ca²⁺]_i and cardiac contractility within membrane regions (T-tubules) in close proximity to the Ca²⁺ regulatory machinery (e.g. L-type Ca²⁺ channels, sarcoplasmic reticulum Ca stores, NCX) and ₁ localises to the surface sarcolemma and plays a general housekeeping role by regulating bulk [Na⁺]_i [22]. In agreement with this concept, selective inhibition of ₂ activity in mouse astrocytes with genetically modified levels of ₂ subunit expression, increases [Na⁺]_i and [Ca²⁺]_i (via NCX) in the cytosolic environment between plasma (PM) and endoplasmic reticulum (ER) membranes [16].

Although the validity of the compartmentalisation model has recently been contested [11,37], immunofluorescence studies in guinea-pig ventricular cardiac myocytes suggest that ₁ subunits are predominantly located in the peripheral sarcolemmal whilst ₂ are mainly distributed in the T-tubules [42]. A similar pattern has been reported in primary cultured rat astrocytes, neurons and arterial myocytes [24], but the opposite pattern has been reported in rat ventricular myocytes [29]. Further studies are required to clarify this situation.

Myocyte detubulation enables direct functional measurements of ion channel and transporter function in surface sarcolemma (SSL) vs. T-tubule membranes [3,25]. Detubulation is achieved by subjecting myocytes to osmotic shock which seals off the T-tubules leaving them functionally intact but isolated from the SSL. In detubulated myocytes only currents carried by SSL channels and transporters are accessible. By this method it has been demonstrated that L-type Ca²⁺ current (I_Ca) [25], NCX activity, and Na⁺/K⁺-ATPase activity [8,47] are preferentially concentrated in the T-tubules of rat ventricular myocytes. This evidence is in favour of a model whereby all the components required for efficient excitation–contraction coupling are localised in the T-tubules and in close proximity to the SR Ca²⁺ store. However, detubulation has yet to be used to investigate the distribution of Na⁺/K⁺-ATPase ₁ and ₂-subunit function in T-tubule and SSL membranes. This may shed light on different physiological roles of ₁ and ₂ in the heart.

In the present study we have assessed the functional distribution of ₁ and ₂ subunits in T-tubule versus SSL membranes by formamide-induced detubulation of mouse ventricular myocytes and measurement of Na⁺/K⁺ pump current (I_pump) and Na⁺/K⁺-ATPase-mediated Na⁺ efflux rate (–d[Na⁺]_i/dt). We have estimated, (i) the composition of plasma membrane surface area in terms of T-tubule and SSL membranes, (ii) I_pump amplitude and density in T-tubule and SSL membranes, (iii) the contribution of I₁ and I₂ to total I_pump, (iv) the I₁:I₂ ratio in T-tubule and SSL membrane compartments, and (v) the effect of ISO stimulation on ₁ and ₂ Na⁺/K⁺-ATPase activity.

	2. Materials and methods

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
2.1. Animals
All animals used in this study received humane care in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institute of Health (NIH Publication No. 85-23, revised 1996). This study was also subjected to local ethical review by the Ethical Review Process Committee of King's College London and Loyola University Chicago.

2.2. Myocyte isolation
C57Bl/6 mice were anaesthetized by intraperitoneal injection of sodium pentobarbitone (60 mg/kg) and heparin (100 IU). Hearts were excised, perfused in a retrograde fashion through Langendorff apparatus. Ventricular myocytes were isolated by Langendorff perfusion and enzymatic dispersion (0.3 mg/mL Type-2 collagenase, Worthington) following a modified version of the AfCS procedure protocol (# PP00000125) [21].

2.3. Myocyte detubulation
Detubulation was induced by osmotic shock as described previously [25]. Briefly, 1.5 mol/L formamide was added to the cell suspension for 15–20 min, then withdrawn. Myocytes were plated on laminin-coated coverslips for 20 min and incubated with 10 µmol/L di-8-aminoaphthylethenylpyridinium (di-8-ANEPPS) in Ca²⁺-free Tyrode solution (in mmol/L, NaCl 137, KCl 5.4, MgCl₂ 0.5, glucose 10, HEPES 10, pH 7.4) in the dark for 20 min, imaged by confocal microscopy (excitation 488 nm, peak emission 515+/–15 nm) and analysed with Image J software (NIH). All confocal images were captured midway through the myocyte z-axis.

2.4. Electrophysiological recording of Na⁺/K⁺ pump current (I_pump)
Mouse ventricular cardiac myocytes were voltage-clamped and whole-cell I_pump recorded at 35 °C using the perforated-patch technique. Electrodes were made from thin-walled (1.5 mm outer diameter, 1.17 mm inner diameter) borosilicate glass capillaries (Harvard Apparatus Ltd, UK) and fire-polished using a three-stage electrode puller (DMG Universal Puller, Zeitz-Instrumente Vetriebs GMBH, Germany). Electrode resistance was 1–2 M when filled with the standard pipette solution. Following gigaohm seal formation, series resistance was monitored with a repetitive 5 mV pulse (–80 mV holding potential). During membrane permeabilization, series resistance typically fell to 10–15 M within 10 min. Membrane capacitance was recorded after permeabilization by standard techniques [4] by imposing a 25 ms square step from –80 to –75 mV and integrating the area under the capacitance transient. I_pump was recorded continuously at 10 Hz sampling frequency at 0 mV. Pipette and extracellular solutions were designed to inhibit all voltage-gated channels and the Na/Ca exchanger. Standard pipette solution contained (in mmol/L) CsCH₃O₃S 90, NaCH₃O₃S 35, NaCl 15, CsCl 5, MgCl₂ 1, HEPES 10, pH 7.2 at 35 °C with CsOH. Amphotericin B (225 µg/mL) (from Streptomyces, Sigma, UK) in DMSO (0.74% v/v) was added to the pipette solution on the day of use. Standard K-containing extracellular solution (5K) contained (in mmol/L) NaCl 140, KCl 5, MgCl₂ 1, NiCl₂ 2, BaCl₂ 1, procaine 0.5, glucose 10, HEPES 10, pH 7.4 at 35 °C. K-free solution (0K) was prepared by removing KCl with no correction for osmolarity. In all experiments I_pump was defined as that sensitive to the removal of extracellular K and was calculated as the product of steady-state 5K minus 0K current. Ouabain was added to 5K solution on the day of use (from a 10 mmol/L stock) and protected from light.

2.5. Measurement of Na⁺ efflux through the Na⁺/K⁺ pump
Na/K-pump flux was determined as the rate of pump-mediated [Na⁺]_i decline and dual excitation fluorescence measurements (at 340 and 380 nm; F340 and F380) were performed as previously described [9]. Myocytes were Na⁺-loaded by inhibiting the Na⁺/K⁺ pump in a K⁺-free solution containing (mmol/L): 145 NaCl, 2 EGTA, 10 HEPES, and 10 glucose (pH=7.4). [Na⁺]_i decline was measured on pump reactivation in solution containing (mmol/L): 140 TEA-Cl, 4 KCl, 2 EGTA, 1 MgCl₂, 10 HEPES, and 10 glucose (pH=7.4). Because cell volume does not change with this protocol [10], [Na⁺]_i decline reflects Na⁺ efflux. The rate of [Na⁺]_i decline (–d[Na⁺]_i/dt) was plotted versus [Na⁺]_i and fitted with: –d[Na⁺]_i/dt=V_max/(1+(K_m/[Na⁺]_i)^nHill). In separate experiments, –d[Na⁺]_i/dt was measured in the presence of 10 mmol/L ouabain to determine Na⁺ pump independent Na⁺ efflux. This was subtracted from Na⁺/K⁺ pump-mediated efflux. In some experiments, cells were treated with 1 µmol/L ISO during the latter part of pump inhibition and throughout reactivation. Ouabain (10 µmol/L) was used in some experiments to preferentially inhibit high ouabain affinity Na⁺/K⁺-ATPase ₂ subunits. All experiments measuring [Na⁺]_i with SBFI were carried out at room temperature (25 °C).

2.6. Statistical analysis
Quantitative data are shown as mean±standard error of the mean (SEM). n values for electrophysiological experiments are given as the number of cells from number of animals. Student t test was used for statistical discriminations, with P<0.05 considered significant and non-significance indicated (ns).

	3. Results

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
3.1. Formamide treatment induces detubulation of mouse ventricular myocytes
Fig. 1A shows an x–y confocal image of a control myocyte stained with di-8-ANEPPS (representative of the staining pattern in 30 cells from 7 hearts): the T-tubule membrane network is clearly visible. The dotted line running along the longitudinal axis of the cell marks the area of fluorescence intensity (FI) analysis and shows repetitive fluorescence peaks at 2 µm intervals and two larger peaks representing SSL membrane staining. Fast Fourier transformation (FFT) of FI profiles allows determination of T-tubule interval. FFT of FI profiles from control myocytes (13 cells from 6 hearts) gave a prominent peak at 0.54±0.01 µm^–1 i.e., a mean T-tubule interval of 1.84±0.02 µm. Fig. 1B is a representative (54 cells from 7 hearts) x–y confocal image of a di-8-ANEPPS stained detubulated myocyte. FFT of FI profiles from detubulated myocytes reveals no periodicity. These results are indicative of successful detubulation and limited dye access from the SSL to the T-tubule membrane network in detubulated myocytes.

View larger version (27K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 1 The effect of detubulation on T-tubule structure in mouse ventricular myocytes. Representative x–y confocal images (captured midway through the depth of the myocyte) of a control (A) and detubulated (B) mouse ventricular cardiac myocyte stained with di-8-ANEPPS. Images are representative of the staining pattern observed in 30 cells from 7 hearts and 54 cells from 7 hearts respectively. The fluorescence intensity (FI) profiles (arbitrary units) beneath the images relate to the region of membrane marked by the dotted red line. Fast Fourier transformation (FFT) of FI profiles from control myocytes (13 cells from 6 hearts) gave a mean inter-peak interval of 0.54±0.01 µm–1 i.e., 1.84±0.02 µm. No prominent peak was observed in FFT of FI profiles from detubulated myocytes.

 
3.2. Cell capacitance and I_pump distribution in surface sarcolemmal and T-tubular membrane compartments
In cardiac myocytes the membrane system is composed of surface sarcolemmal (SSL) and T-tubular compartments. T-tubule cell capacitance and localised Na⁺/K⁺ pump current were calculated by subtracting control values from those recorded in detubulated myocytes (T-tubule=Total–SSL). Membrane capacitance was reduced from 181±12 pF in control to 126±17 pF in detubulated myocytes. These data demonstrate that membrane surface area is composed of 30% T-tubule (54 pF) and 70% SSL (126±17 pF) membranes. I_pump amplitude recorded at 0 mV in 5 mmol/L [K]_o and 50 mmol/L [Na⁺]_i was reduced from 257±22 pA in control myocytes (1.42±0.1 pA/pF) to 151±17 pA in detubulated myocytes (1.20±0.04 pA/pF). Therefore, although T-tubules represent only 30% of the membrane surface area, Na⁺ pumps residing there generate 41% (106 pA) of total I_pump (the remaining 59% is generated in the SSL). These data are summarised in Fig. 2. Normalising I_pump amplitude data to cell capacitance demonstrates that functional I_pump density in T-tubular membranes (1.94 pA/pF) is 60% higher than in SSL membranes (1.20 pA/pF) (T-tubule:SSL I_pump density ratio=1.6:1).

View larger version (11K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 2 Cell capacitance and Ipump amplitude/density in SSL and T-tubule membranes recorded in control and detubulated myocytes. (A) Cell capacitance was reduced from 181±12 pF in control (n=28) to 127±17 pF in detubulated (n=7) myocytes. Hence 70% of total membrane surface area is localised in the SSL compartment (SSL capacitance=127±17 pF) and the remaining 30% is localised in the T-tubules (calculated T-tubule capacitance=54 pF). (B) Average Ipump amplitude was 257±22 pA in control (n=18 cells, 7 hearts) and 151±17 pA in detubulated (n=5 cells, 4 hearts) myocytes. Hence 151±17 pA (59%) of total recordable Ipump is generated by Na+ pumps residing in SSL membranes and the remaining 106 pA (41%) by those residing in the T-tubules. (C) Total functional Ipump density was 1.42±0.1 pA/pF in control cells, 1.20±0.04 pA/pF in detubulated cells (SSL) and 1.94 pA/pF (calculated) in the T-tubules.

 
We also measured the rate of Na⁺/K⁺-ATPase-mediated Na⁺ efflux (–d[Na⁺]_i/dt) in control and detubulated myocytes (Fig. 3). Maximal Na⁺ efflux rate (V_max) was 10.7±1.9 mmol/min in control myocytes and 7.5±0.9 mmol/min following detubulation. Despite this not achieving the level of statistical significance, this 30% difference in the mean V_max values is not incompatible with the suggestion from the voltage-clamp data that T-tubular Na⁺/K⁺-ATPase activity accounts for about 40% of the total cellular Na⁺ efflux.

View larger version (23K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 3 Na+/K+-ATPase-mediated Na+ efflux in control and detubulated myocytes plotted as a function of intracellular Na+ concentration ([Na+]i). The rate of [Na+]i decline (–d[Na+]i/dt) was measured in control (n=5 cells, 4 hearts) and detubulated (n=4 cells, 3 hearts) myocytes. Averaged data was fitted with a Hill equation and Vmax, Km and Hill slope (h) estimated for each data set. Maximal Na+ efflux rate (Vmax) was reduced 10.7±1.9 mmol/min in control myocytes and 7.5±0.9 mmol/min following detubulation (ns). The Km and slopes of the fitted curves were estimated in control cells to be 16.6±1.3 mmol/L, and 2.4±0.1 and, in detubulated cells, to be 13.8±3.0 mmol/L (ns) and 2.3±0.1 (ns) respectively.

 
3.3. I_pump composition in control myocytes (SSL and T-tubule membranes)
The contribution of I₁ and I₂ to total I_pump was defined by investigating the dose-dependent inhibition of I_pump with ouabain. I₁ (low-affinity) and I₂ (high-affinity) were defined by their differing sensitivity to ouabain. Maximal I_pump inhibition was achieved by exposure to 10 mmol/L ouabain or 0K solution. Recovery from inhibition was rapid and complete with a return to the pre-inhibition level within 3 min. Fig. 4A is a current recording demonstrating dose-dependent inhibition of I_pump with ouabain. Average data representing the percentage inhibition of I_pump by ouabain was fit with a two-site binding hyperbolic function and I₁ and I₂ determined by curve stripping (Fig. 4B). In control myocytes I₁ contributed 88% to the total recordable current with a K_d for ouabain of 105 µmol/L and I₂ contributed the remaining 12% with a K_d of 0.3 µmol/L.

View larger version (12K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 4 (A) Current recording from an experimental protocol investigating the dose-dependent inhibition of Ipump with ouabain in control myocytes (mathematical curve stripping of I1 and I2 components). (B) Average data (n=5 for each [ouabain]) was fitted with a hyperbolic two-site binding function Y=Bmax1*X/(Kd1+X)+(100–Bmax1)*x/(Kd2+X) to represent I1 (low affinity) and I2 (high affinity) Ipump components. Dashed curves represent I1 (Bmax1 88%, Kd1 104.8 µmol/L) and I2 (Bmax2 12%, Kd2 0.3 µmol/L).

 
To support the above I_pump data, Na⁺/K⁺-ATPase-mediated Na⁺ efflux was recorded in the presence of low dose ouabain (10 µmol/L) to preferentially inhibit Na⁺/K⁺-ATPase ₂ subunits. Curve stripping analysis of our I_pump data (Fig. 4B) suggests that 10 µmol/L ouabain, will inhibit 97% of ₂-mediated Na⁺/K⁺-ATPase activity and only 9% ₁ activity. Under these conditions ₁-mediated Na⁺ efflux predominates due to 11-fold specificity for ₂ inhibition. ₂-mediated Na⁺/K⁺-ATPase activity was calculated as the difference between total and ₁-mediated activity. Fig. 5 represents total Na⁺/K⁺-ATPase-mediated Na⁺ efflux, and that mediated via ₁ and ₂ subunits. Curve fitting with the Hill equation demonstrated that maximal Na⁺ efflux rate (V_max) was 10.7±1.9 mmol/min in control myocytes. 80% of this Na⁺ efflux capacity was due to ₁ subunits (8.6±1.6 mmol/min) and the remaining 20% via ₂. These data correlate well with estimates of I_pump composition in terms of I₁ and I₂. Additionally, these data suggest that ₁ and ₂ subunits have very similar affinity for Na⁺ ions, with K_m values of 16.6±0.8 and 16.7±2.6 mmol/L respectively (ns).

View larger version (24K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 5 Total and 1- and 2-mediated Na+ efflux. Total Na+ efflux (n=5 cells, 4 hearts) was determined under control conditions (Vmax=10.7±1.9 mmol/min, Km=16.6±1.2 mmol/L and h=2.3±0.1). 1-mediated Na+ efflux (n=5 cells, 4 hearts) was defined as that resistant to 10 µmol/L ouabain (Vmax=8.6±1.6 mmol/min, Km=16.6±0.8 mmol/L, h=2.3±0.1). 2-mediated Na+ efflux was derived mathematically (Total Na+ efflux- 1-mediated Na+ efflux) (Vmax 2.1±3.1 mmol/min, Km 16.7±2.6 mmol/min, h=4.1±0.8). Curves were fitted and Vmax, Km and n values derived as described in the legend to Fig. 3. (Error bars for 2 data have been excluded for simplicity).

 
3.4. I₁ and I₂ amplitude and density in SSL and T-tubular membranes
Having determined the contribution of ₁ and ₂ subunits to I_pump in control myocytes we constructed a ouabain dose–response curve in detubulated myocytes. Under these conditions, I₁ contributes 94% and I₂ only 6% to total recordable I_pump (K_d for ouabain of 170 and 0.2 µmol/L respectively) (Fig. 6). These data define the percentage composition of total I_pump, in terms of I₁ and I₂ in the SSL membrane compartment.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 6 Dose dependent inhibition of Ipump with ouabain in detubulated myocytes. I1 and I2 components were derived by mathematical curve stripping after fitting average data with a hyperbolic two-site binding function Y=Bmax1*X/(Kd1+X)+(100–Bmax1)*x/(Kd2+X). In detubulated myocytes, I1 contributes 93.8% (Kd 170.3 µmol/L) and I1 contributes 6.2% (Kd 0.19 µmol/L) to total recordable Ipump.

 
In control myocytes cell capacitance was 181 pF and total I_pump amplitude (257 pA) was composed of 88% I₁ (226 pA) and 12% I₂ (31.1 pA). Therefore, I₁ density was 1.25 pA/pF, and I₂ density was 0.17 pA/pF. In detubulated myocytes (in which only SSL membranes contribute to cell capacitance and only SSL pumps contribute to whole-cell I_pump), total I_pump amplitude (151 pA) was composed of 94% I₁ (142 pA) and 6% I₂ (9.4 pA). After normalising for SSL cell capacitance (127 pF), SSL I₁ density was 1.12 pA/pF and I₂ density was 0.07 pA/pF. With the above information, T-tubule I₁ and I₂ amplitude (84 pA and 22 pA respectively) and density (1.54 pA/pF and 0.39 pA/pF respectively) can be determined mathematically. These data are summarised in Table 1, panel A.

View this table: [in this window] [in a new window]   Table 1A 1 and I2 in SSL and T-tubule membrane compartments

 
3.5. β-adrenergic regulation of Na⁺/K⁺-ATPase ₁ and ₂ subunits
In addition to investigating whether Na⁺/K⁺-ATPase -subunit isoforms demonstrate differential subcellular localisation, we also determined whether this is associated with differential regulation by PKA. 1 µmol/L ISO was used to maximally activate the protein kinase A (PKA) signalling cascade and mimic the effect of β-adrenergic stimulation. Under control conditions, ISO induced a significant (P=0.035) 26% increase in Na⁺ sensitivity, shifting the K_m for Na⁺ (K_1/2 Na⁺) from 16.6±1.2 mmol/L to 12.3±1.3 mmol/L but had no effect on V_max (from 10.7±1.9 to 11.2±1.7 mmol/min) (Fig. 7A). In the presence of 10 µmol/L ouabain, ISO induced a similarly significant (P=0.048) 20% stimulation of ₁-mediated Na⁺/K⁺-ATPase Na⁺ affinity (K_m decreasing from 16.6±0.8 to 13.3±1.4 mmol/L) again with no effect on V_max (from 8.6±1.6 to 9.3±0.7 mmol/min) (Fig. 7B). ₂-mediated Na⁺/K⁺-ATPase activity was calculated mathematically as previously described (Fig. 7C). ISO had no effect on ₂-mediated V_max (from 2.1±3.1 to 1.8±2.9 mmol/min) and Na⁺ affinity (16.7±2.6 to 11.2±3.4 mmol/L). It is clear from the data in Fig. 7C that the error associated with ₂-mediated Na⁺/K⁺-ATPase activity is very large. These data are derived mathematically and ₂ represents only a small component of total Na⁺/K⁺-ATPase activity.

View larger version (18K): [in this window] [in a new window] [Download PowerPoint slide]   Fig. 7 The effect of β-adrenergic stimulation on Na+/K+-ATPase-mediated Na+ efflux. (A) Influence of ISO (1 µmol/L) on total Na+ efflux (n=8 cells, 5 hearts) measured in intact myocytes. Control data from Fig. 5 is included for comparison (see legend to Fig. 5 for control data). ISO did not affect Vmax or Hill slope (Vmax=11.2±1.7 mmol/min), h=2.6±0.1). ISO, however, significantly decreased the Km for Na+ activation to 12.3±1.3 mmol/L (P<0.05). (B). Influence of ISO on 1-mediated Na+ efflux (9 cells, 5 hearts). ISO did not affect Vmax or Hill slope (Vmax=11.2±1.7 mmol/min, h=2.3±0.1). ISO, however, significantly decreased the Km for Na+ activation to 12.3±1.3 mmol/L (P<0.05). (C) Influence of ISO on 2-mediated Na+ efflux. ISO did not affect Vmax or Hill slope (Vmax=1.8±2.9 mmol/min, h=4.1±0.8). The Km for Na+ activation was not significantly different to that for control (Km=11.2±3.4 mmol/L) (ns). Curves were fitted as described in the legend of Fig. 3 and Vmax, Km and h values derived.

 

	4. Discussion

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 
In the present study we have investigated the distribution and function of Na⁺/K⁺-ATPase ₁ and ₂-subunits in SSL and T-tubule membranes in mouse ventricular cardiac myocytes using the technique of myocyte detubulation.

The accuracy of this experimental approach relies on successful and efficient detubulation. The close correlation between the reduction in cell surface area reported here (30%) and direct electron microscopic measurements of total sarcolemma in T-tubules in mouse ventricular myocytes (36%) [33] suggests that this method is quantitatively reliable. Furthermore, staining of control myocytes with di-8-ANEPPS revealed a mean T-tubule interval of 1.84 µm, which is very similar to that observed in the rat (1.86 µm) [3]. Repetitive T-tubular staining was completely lost following detubulation.

Although ₁ and ₂ isoforms are present in mouse ventricular myocytes, ₁ is the predominant Na⁺/K⁺-ATPase isoform. ₁-mediated I_pump (I₁) contributes 88% to total recordable I_pump and ₂-mediated I_pump (I₂) contributes the remaining 12%. The overall subcellular localisation of Na⁺/K⁺-ATPase activity indicates that I_pump density is 60% higher in T-tubule vs. SSL membranes (although T-tubule membranes represent only 30% of total membrane area) and that Na⁺ pumps residing there generate 41% of total I_pump; 37% of I₁ and 70% of I₂. Moreover, this study provides the first quantitative determination of the relative distribution of ₁ and ₂-mediated Na⁺/K⁺-ATPase activity in mouse ventricular myocytes. We have shown that I₁ density predominates over I₂ in both SSL and T-tubule membrane compartments. However, the relative ratio of I₁:I₂ is markedly different in T-tubule versus SSL membranes. I₁ density is substantially higher than I₂ in SSL membranes (I₁:I₂ density ratio of 16:1), but in T-tubule, the dominance of I₁ over I₂ is markedly reduced (4:1). Furthermore the T-tubule:SSL I₁ ratio (1.4:1) suggests that I₁ is relatively uniformly distributed between T-tubule and SSL membranes whereas I₂ is 5 times more concentrated in the T-tubules (T-tubule:SSL ratio of 5.3:1). These data are summarised in Table 1B. A similar pattern has been reported in rat ventricular myocytes in a recent abstract, with 4.5 times higher functional density of I₂ in the T-tubules and uniform I₁ distribution between T-tubule and SSL membranes [6].

View this table: [in this window] [in a new window]   Table 1B Relative ratios

 
Recent studies in detubulated rat myocytes have reported that I_Ca, NCX and Na⁺/K⁺-ATPase activity are concentrated in the T-tubules [8,25,47]. Hence, co-localisation of a specific Na⁺/K⁺-ATPase subunit isoform with NCX and the L-type Ca channel in T-tubules would form a structural basis of cardiac excitation–contraction coupling and local control of contractility as described by the compartmentalisation model of James et al. [22]. Recently, the validity of this compartmentalisation model has been contested [11,37], and the authors have now concluded that both ₁ and ₂ isoforms can indirectly regulate cardiac contractility through modulation of forward mode Na/Ca exchange. Our data suggests that ₁ and ₂ subunits are differentially localised. I₁ is greater than I₂ in both T-tubule and SSL membrane compartments but the predominance of I₁ over I₂ is lower in T-tubule. Therefore, it is possible that by altering the extent to which ₁ predominates over ₂ (i.e. altering the I₁:I₂ balance), previously hidden differential physiological roles for the two isoforms may be revealed.

In addition to differential subcellular localisation we have also demonstrated that ISO significantly stimulates ₁-mediated Na⁺/K⁺-ATPase activity (predominantly via an increase in Na⁺/K⁺-ATPase Na⁺ affinity). In agreement, recent data from our laboratory have demonstrated isoform-specific stimulation of I₁ in guinea-pig myocytes following PKA stimulation with forskolin [42]. Conversely, in the present study we have shown that ISO has no significant effect on ₂-mediated Na⁺/K⁺-ATPase activity, but due to the small contribution of ₂ to total Na⁺/K⁺-ATPase activity coupled with its mathematical derivation, this conclusion should be viewed with caution. Previously published studies from ourselves [42] and others [15] have also suggested that β-stimulation activates ₁ but not ₂. However, on the basis of the data presented in this present study, it is possible that ISO does influence ₂-mediated pump function but this is below the limit of detection of this method.

In terms of β-adrenergic stimulation in the heart, many proteins involved in excitation–contraction coupling are targets for PKA phosphorylation (e.g. L-type Ca²⁺ channel, phospholamban, ryanodine receptor, troponin I). Until recently the exact mechanism of Na⁺/K⁺ pump regulation by PKA has remained elusive. It is now clear that this role is played by phospholemman (PLM) [1,7,42], a member of the FXYD family of proteins that are tissue specific regulators of the Na⁺/K⁺ pump. PLM is the primary sarcolemmal substrate for PKA [35] and PKC [36] and regulates the cardiac Na⁺/K⁺ pump by applying a tonic inhibition that is relieved by genetic PLM knockout and PLM phosphorylation [1,7]. Previously, we have demonstrated both functional and physical association between PLM and the Na⁺/K⁺-ATPase ₁ isoform [42] (but not with ₂), and more recently we have demonstrated that the stimulatory effect of ISO on I_pump in PLM wildtype voltage-clamped mouse ventricular myocytes is preferentially mediated via stimulation of ₁-mediated current [1]. With regards to biochemical evidence, co-immunoprecipitation studies have demonstrated association of PLM with the ₂ subunit in rabbit ventricular myocytes and bovine sarcolemmal microsomes [2,5]. However no association has been reported in guinea-pig ventricular myocytes and rat cardiac homogenates [13,42].

The evidence presented here supports a model whereby ₁ and ₂ subunits demonstrate differential localisation and potentially differential regulation by PKA/β-adrenergic stimulation in mouse ventricular T-tubule and SSL membranes. As β-stimulation leads to elevated [Na⁺]_i as a direct consequence of positive chronotropy [12,18,43], it may be that the ₁ subunit, located in SSL membranes and regulated by PLM, is primarily involved in controlling bulk [Na⁺]_i (as suggested by James et al. [22]) and controlling the delicate balance of [Na⁺]_i by allowing it to rise sufficiently to contribute to the positive inotropic effect of β-stimulation while protecting against the deleterious effects of [Na⁺]_i and [Ca²⁺]_i overload which may lead to cardiac arrhythmias and diastolic dysfunction.

	Acknowledgements

 
This work was supported by grants from the Medical Research Council, the British Heart Foundation and the University of London Central Research Fund.

	Notes

 
Time for primary review 39 days

	References

Top Abstract 1. Introduction 2. Materials and methods 3. Results 4. Discussion References

 

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