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VASCULAR BIOLOGY

Functional analysis of Na⁺/K⁺-ATPase isoform distribution in rat ventricular myocytes

Department of Physiology, Loyola University Chicago, Maywood, Illinois

Submitted 30 November 2006 ; accepted in final form 28 March 2007

	ABSTRACT

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The Na⁺/K⁺-ATPase (NKA) is the main route for Na⁺ extrusion from cardiac myocytes. Different NKA -subunit isoforms are present in the heart. NKA-1 is predominant, although there is a variable amount of NKA-2 in adult ventricular myocytes of most species. It has been proposed that NKA-2 is localized mainly in T-tubules (TT), where it could regulate local Na⁺/Ca²⁺ exchange and thus cardiac myocyte Ca²⁺. However, there is controversy as to where NKA-1 vs. NKA-2 are localized in ventricular myocytes. Here, we assess the TT vs. external sarcolemma (ESL) distribution functionally using formamide-induced detubulation of rat ventricular myocytes, NKA current (I_Pump) measurements and the different ouabain sensitivity of NKA-1 (low) and NKA-2 (high) in rat heart. Ouabain-dependent I_Pump inhibition in control myocytes indicates a high-affinity NKA isoform (NKA-2, K_1/2 = 0.38 ± 0.16 µM) that accounts for 29.5 ± 1.3% of I_Pump and a low-affinity isoform (NKA-1, K_1/2 = 141 ± 17 µM) that accounts for 70.5% of I_Pump. Detubulation decreased cell capacitance from 164 ± 6 to 120 ± 8 pF and reduced I_Pump density from 1.24 ± 0.05 to 1.02 ± 0.05 pA/pF, indicating that the functional density of NKA is significantly higher in TT vs. ESL. In detubulated myocytes, NKA-2 accounted for only 18.2 ± 1.1% of I_Pump. Thus, 63% of I_Pump generated by NKA-2 is from the TT (although TT are only 27% of the total sarcolemma), and the NKA-2/NKA-1 ratio in TT is significantly higher than in the ESL. The functional density of NKA-2 is 4.5 times higher in the T-tubules vs. ESL, whereas NKA-1 is almost uniformly distributed between the TT and ESL.

T-tubules; Na⁺/K⁺ pump current; ouabain; external sarcolemma; detubulation

The NKA has two essential subunits: and . The subunit (110 kDa) contains the binding sites for Na⁺-K⁺-ATP and cardiac glycosides. The smaller subunit (50 kDa) has a role in the processing and in the proper membrane insertion of the pump. There are three (1–3) and three (1–3) NKA subunit isoforms in the heart, with any - combination resulting in a functional pump. NKA-1 is present in the heart of all species studied, whereas the expression of NKA-2 and NKA-3 differs significantly between species (16). For instance, the adult rodent heart expresses NKA-1 and NKA-2, whereas dogs and monkeys do not have the NKA-2 subunit (11). In humans, all three NKA- isoforms can be detected (22).

It has been suggested that different NKA isoforms may function differently in the cell, depending on differential localization in the membrane. Juhaszova and Blaustein (14) showed that in smooth muscle NKA-2 is localized at the junctions with the sarcoplasmic reticulum (SR) and regulates local Na⁺/Ca²⁺ exchange and [Ca²⁺]_i. James et al. (13) showed that mouse hearts with genetically reduced levels of NKA- were hypercontractile as a result of increased Ca²⁺ transients during the contractile cycle. In contrast, hearts with reduced levels of NKA-1 were hypocontractile. Moreover, inhibition of NKA- with ouabain increased the contractility of heterozygous NKA-1 hearts. These data indirectly implicate a selective involvement of NKA-2 in cardiac myocyte Ca²⁺ regulation. Further support for this idea came from the observation that increased expression of NKA-2 decreased I_NCX and Ca²⁺ transients in mouse ventricular myocytes (24). Dostanic et al. (8) showed that replacing mouse NKA-2 with a low-ouabain-affinity mutant leads to a loss of glycoside inotropy, which suggests that NKA-2 regulates local [Ca²⁺]_i near the Na⁺/Ca²⁺ exchange, as in smooth muscle. However, the same authors later showed that NKA-1 is also functionally and physically coupled with NCX in cardiac myocytes (9).

Immunofluorescence data indicated that NKA is located in both external sarcolemma (ESL) and T-tubules (TT) in ventricular myocytes (16, 21). The differential NKA-1/NKA-2 localization (TT vs. ESL) in cardiac cells is controversial. In the rat, McDonough et al. (16) found the NKA-1 isoform to be preferentially distributed in the TT, while NKA-2 was uniformly distributed in the TT and peripheral sarcolemma. Conversely, Silverman et al. (21) showed that in guinea pig ventricular myocytes NKA-2 is mainly in TT and NKA-1 is predominantly on the external sarcolemma. However, these are mostly qualitative data, limited by the selectivity of the antibodies and do not give information about the function of NKA isoforms in the TT vs. ESL.

Kawai et al. (15) developed a novel method to detubulate ventricular myocytes using formamide. Upon withdrawal of 1.5 M formamide, the TT seal off, and only currents carried by ESL channels and transporters are accessible. Notably, this procedure has no effect on ionic currents measured in atrial myocytes, which lack endogenous TT, indicating that formamide exposure and withdrawal do not by themselves alter ion channel function (3). We have previously used this method and Na⁺/K⁺ pump current (I_Pump) measurements in control and detubulated rat and mouse ventricular myocytes to show that the NKA functional density is significantly higher in the TT vs. ESL (1, 6).

The aim of this paper is to investigate the functional distribution of NKA-1 and NKA-2 in rat between the TT and ESL. In rat ventricular myocytes, we can distinguish the I_Pump generated by NKA-1 and NKA-2 based on their different ouabain affinities: NKA-2 is ouabain sensitive, whereas NKA-1 is ouabain resistant (12). We can also measure I_Pump due to whole cell NKA (measurements in control myocytes) and due to NKA in the ESL (measurements in detubulated myocytes). Obviously, the difference is the I_Pump in the TT. Here, we measured the dose-dependent I_Pump inhibition by ouabain in control and detubulated rat ventricular myocytes. Combining these approaches allowed us to directly assess what fraction of the external sarcolemmal I_Pump is due to NKA-1 vs. NKA-2 and likewise for the TT.

	MATERIALS AND METHODS

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Myocytes isolation and detubulation procedure. The procedure for isolation of rat ventricular myocytes has been described previously (7) and was approved by the Loyola University (Chicago, IL) animal welfare committee. Briefly, rats were anesthetized by intraperitoneal injection of nembutal (1 mg/g). Hearts were excised quickly and placed on a Langendorff perfusion apparatus. Hearts were perfused for 5–7 min with nominally Ca²⁺-free Tyrode's solution, then perfusion proceeded with added collagenase (1 mg/ml) and albumin (0.05%). When the heart became flaccid, the atrial and left ventricular tissues were excised and cut into small pieces for further incubation (5–10 min, with 0.4 mg/ml collagenase for the ventricular tissue and 12–20 min in the presence of 0.4 mg/ml collagenase and 0.2 mg/ml protease for the atria). The tissue was then filtered and Ca²⁺ concentration in the cell suspension was progressively increased to 1 mM. The standard Tyrode's solution used in these experiments contained (in mM): 140 NaCl, 4 KCl, 1 MgCl₂, 10 glucose, 5 HEPES and 1 CaCl₂ (pH = 7.4). All experiments were done at room temperature (23–25°C).

Detubulation was induced by osmotic shock, as described previously (6, 15). Briefly, 1.5 M formamide was added to the cell suspension for 15–20 min; then the cells were returned to the standard solution. Upon formamide withdrawal, the T-tubules seal off and lose electrical contact with sarcolemma.

Na⁺/K⁺ pump current measurements. Isolated ventricular and atrial myocytes were plated on laminin-coated glass coverslips and pretreated with 1 µM thapsigargin for 10 min. Control and detubulated myocytes were whole cell voltage-clamped using patch electrodes made from borosilicate glass capillaries, as previously described (6). When filled with the standard pipette solution, electrode resistance was 1.5–2.5 M. Current signals were recorded using an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). Membrane capacitance (C_m) and series resistance were calculated from a 5-mV voltage step from a holding potential of –70 mV. The pipette solution contained (in mM): 30 NaCl, 70 NaOH, 70 aspartic acid, 20 K⁺-aspartate, 20 TEA-Cl, 10 HEPES, 5 Mg-ATP, 0.7 MgCl₂ (1 mM free Mg), 3 BAPTA, 1.15 CaCl₂ (100 nM free Ca²⁺), pH = 7.2. I_pump was activated in whole cell voltage-clamp by rapid switch from 0 to 4 mM external K⁺ at a holding potential of –20 mV. We have shown previously that the K⁺-activated current is ouabain sensitive (5). The external solution contained (in mM): 136 NaCl, 5 NiCl₂, 2 BaCl₂, 1 MgCl₂, 5 HEPES, 5 glucose, ± 4 KCl, pH = 7.4.

Dose-dependent I_Pump inhibition by ouabain. After reaching the whole cell configuration, myocytes were held at –70 mV in K⁺-free external solution for at least 8 min, to allow equilibration of intracellular and pipette Na⁺. Then, I_Pump was activated at –20 mV by switching to 4 mM K⁺ external solution. Under these conditions, about 75% of the pumps is activated [assuming a K_1/2 for external K⁺ of 1.5 mM (17)]. We then recorded the current, while adding increasing concentrations of ouabain. For each ouabain concentration, I_Pump was considered at steady state if the holding current was constant for at least 10 s. We also repeated the analysis using exponential extrapolation to idealized steady state at longer times and obtained virtually identical results (not shown). Control experiments (not shown) indicated that I_Pump does not run down significantly during the duration of the experiment. We calculated the percentage of I_Pump inhibition for each ouabain concentration as 100·(I_Pump0 – I_PumpOua)/I_Pump0, where I_Pump0 is I_Pump in the absence of ouabain (i.e., the outward current induced by adding 4 mM K⁺ to the external solution) and I_PumpOua is I_Pump at the respective ouabain concentration. Mean data were fit with a two-binding site equation B₁·[Ouabain]/(K₁ + [Ouabain]) + (100 – B1)·[Ouabain]/(K₂ + [Ouabain]) to derive the K_1/2 for ouabain binding to NKA-1 and NKA-2 and their relative contribution to I_Pump. The fit was done using Origin software (Microcal Software, Northampton, MA) and the best fit was identified by a minimum ².

Statistical analysis. Data are expressed as means ± SE. Unpaired Student's t-test was used for statistical discriminations, with P < 0.05 considered significant.

	RESULTS

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NKA functional density is significantly higher in the TT vs. ESL. The mean membrane capacitance of detubulated myocytes was 120 ± 8 pF (Fig. 1A). Upon detubulation, the TT seal off and lose electrical contact with sarcolemma; thus, C_m measured in detubulated cells is the capacitance of the membrane in the external sarcolemma. In control myocytes, both external sarcolemma and the TT contribute to the measured C_m (164 ± 6 pF). Thus, the difference between control and detubulated myocytes (44 pF) is the C_m of the membrane in the TT (Fig. 1A). These data show that the TT represent about 27% of the total sarcolemma, similar to our previous measurements (6) and electron microscopic morphological data in rat ventricular myocytes (18–20). Also, imaging of detubulated myocytes after staining with membrane-associated fluorescent dyes confirmed lack of TT (not shown). Thus, we are confident that our measurements in detubulated cells reflect almost exclusively external sarcolemma.

View larger version (7K): [in this window] [in a new window]   Fig. 1. Effect of detubulation on the membrane capacitance (A), Na+/K+-ATPase (NKA) current (B) and NKA current density (C). A: Cm decreases from 164 ± 6 pF in control [where both T-tubules (TT) and external sarcolemma (ESL) contribute] to 120 ± 8 pF in detubulated rat myocytes (where only ESL is measured). The difference represents the capacitance of the membrane in the TT. B: IPump decreases from 203 ± 9 pA in control (i.e., IPump produced by NKA present in both TT and ESL) to 122 ± 10 pA in detubulated (due to NKA in ESL only) myocytes. The difference represents IPump originating from the TT. C: IPump density in the entire cell (TT+ESL), ESL, and TT. Data are means for 12 control (7 hearts) and 9 detubulated (5 hearts) myocytes.

NKA-₂ has a larger contribution to I_Pump in control vs. detubulated myocytes. Rat NKA-1 and NKA-2 have distinct ouabain affinities, with NKA-1 having a low affinity and NKA-2 having a high affinity. This allowed us to determine the fraction of I_Pump generated by NKA-1 and NKA-2 by measuring the dose-dependent I_Pump inhibition by ouabain in control and detubulated myocytes. Fig. 2A shows representative traces recorded in a control and a detubulated myocyte. At low concentrations (5 µM), ouabain had a significantly smaller effect in the detubulated cell, whereas at higher concentrations the effect was similar in control and detubulated myocytes.

View larger version (12K): [in this window] [in a new window]   Fig. 2. Dose-dependent IPump inhibition by ouabain in control and detubulated myocytes. A: representative traces in a control and a detubulated myocyte. Note the smaller effect of low ouabain concentrations in the detubulated cell. B: dose-response curves of Ipump inhibition by ouabain in control (10 cells, 7 hearts) and detubulated (8 cells, 5 hearts) rat ventricular myocytes. Data were fit with a two-binding sites equation, indicating the presence of two ouabain binding sites: a high-affinity site, i.e., NKA-2, and a low-affinity site, that is, NKA-1.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Contribution of NKA-1 and NKA-2 to Ipump in control and detubulated rat ventricular myocytes. A: separation of the dose-response curves of Ipump inhibition by ouabain in control and detubulated myocytes into a high- and a low-affinity components. B: K1/2 for the high- and low-affinity components in control and detubulated myocytes. C: relative contribution of NKA-1 and NKA-2 to Ipump in control (TT+ESL) and detubulated (ESL only) myocytes.

On the basis of the contribution of NKA-1 and NKA-2 to I_Pump (Fig. 3C) and the size of I_Pump in control and detubulated myocytes (Fig. 1B), we estimated the relative number of functional NKA-₁ and NKA-₂ in the entire sarcolemma (ESL+TT), ESL and TT (Fig. 4A). We found a larger NKA-₂ amount in TT vs. ESL (despite the smaller surface area of the TT vs. ESL). We then calculated the NKA-₁ and NKA-₂ density (number of pumps/µm²) in the entire sarcolemma (ESL+TT), ESL, and TT (Fig. 4, B and C), assuming that the total pump density in control myocytes is 1,000 pumps/µm² or a maximal turnover rate of 100/s (2). NKA-₁ is rather uniformly distributed between the TT (800 ± 370 /µm²) and ESL (670 ± 70 /µm²), whereas NKA-₂ is 4.5-fold concentrated in the TT (680 ± 120 vs. 150 ± 20/µm² in the external sarcolemma). This estimation of actual NKA-1 vs. NKA-2 site density assumes that the two isoforms have similar turnover rates, i.e., I_pump is proportional to the number of functional NKA molecules (although there are contradictory reports regarding this assumption) (4, 25). If turnover rates do differ between NKA-1 and NKA-2, the relative number of 1 vs. 2-pumps in the TT and ESL would change, but that does not affect our conclusion regarding the functional distribution of NKA-1 and NKA-2 in the TT vs. ESL.

View larger version (12K): [in this window] [in a new window]   Fig. 4. Estimated number (A) and density (B and C) of functional NKA-1 and NKA-2 in the entire sarcolemma (ESL+TT), ESL, and TT. A: we used the ratio between IPump measured in detubulated and control myocytes to calculate the relative number of pumps (including both NKA-1 and NKA-2) in the external sarcolemma. The remaining pumps are located in the T-tubules. Then, we used the relative contribution of NKA-1 and NKA-2 to IPump in control and detubulated myocytes (see Fig. 3C) to determine the number of NKA-1 and NKA-2 in ESL+TT and ESL, respectively. The difference between total sarcolemma and ESL was the number of NKA-1 and NKA-2 in the TT. B: these numbers were divided by the membrane capacity of each component (see Fig. 1A) to derive the pumps density (as % divided by pF) in the ESL+TT, ESL, and TT. The result was converted in terms of pumps/µm2 assuming that the total pump density in control myocytes is 1, 000 pumps/µm2 (2) and 1 µF/cm2. C: cartoon showing the estimated NKA-1 and NKA-2 density in the TT and ESL.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION GRANTS REFERENCES

Here, we investigated the functional distribution of NKA-1 and NKA-2 between the TT and external sarcolemma by taking advantage of 1) the different affinity for ouabain of NKA-1 and NKA-2 in rat and 2) our ability to detubulate ventricular myocytes and thus measure the I_Pump generated by NKA located in the external sarcolemma only. We found that the functional density of NKA-2 is 4.5 times higher in the TT vs. external sarcolemma, whereas NKA-1 is practically uniformly distributed between the TT and ESL (Fig. 4, B and C). However, the amount of NKA-1 and NKA-2 in the TT is comparable (Fig. 4). Our results here for rat are qualitatively similar to our recent results in mouse (in which 12% of total and 6% of surface NKA function was attributable to NKA-2 and that NKA-2 function was 5.3 times higher in T-tubular than surface sarcolemma) (1). So, this distribution difference may be generally true in mammals. A limitation of this study is that detubulation of myocytes may be incomplete. This would underestimate the density of NKA-1 and NKA-2 in the TT. However, we expect this effect to be small (see Ref. 6) and would not change the conclusions of this study.

It has been suggested (13, 14, 24) that different NKA isoforms may have different physiological roles, with NKA-1 acting as a "housekeeper" in maintaining a low [Na⁺]_i and NKA-2 regulating local [Na⁺]_i near NCX and thus cardiac myocyte Ca²⁺ and contractility. In cardiac myocytes, NCX is mostly located in the TT (6, 10) and is preferentially modulated by Ca²⁺ released from the SR, as indicated by functional measurements (23, 26). We found that NKA-2 function is concentrated in the TT vs. external sarcolemma, with the amount of NKA-1 and NKA-2 in the TT being comparable. This raises the intriguing possibility that NKA-2 is localized to sarcolemmal junctions with the SR (48% of the TT area and 8% of ESL in rat ventricle, as measured by electron microscopy) (20) and could be important in regulating local cleft [Na⁺]_i and [Ca²⁺]_i (via NCX) and therefore excitation-contraction coupling. The NKA-1 might predominate in the ESL (92% nonjunctional) and be similar to NKA-2 density in the TT (52% of which is nonjunctional). This hypothesis requires further structural and functional tests in adult ventricular myocytes.

	GRANTS

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This work was supported by grants from the National Institutes of Health Grants HL-30077 and HL-64724 (D. M. Bers).

	ACKNOWLEDGMENTS

 
We thank Jayme O'Brien for myocytes isolation.

	FOOTNOTES

 

Address for reprint requests and other correspondence: D. M. Bers, Dept. of Physiology, Loyola Univ. Chicago, Stritch School of Medicine, 2160 South First Ave., Maywood, IL 60153 (e-mail: dbers{at}lumc.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

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