Phospholemman-Phosphorylation Mediates the -Adrenergic Effects on Na/K Pump Function in Cardiac Myocytes
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循环研究杂志 2005年第8期
the Department of Physiology (S.D., J.B., F.H., K.S.G., D.M.B.), Loyola University Chicago, Maywood, Ill
the University of Virginia (L.J., A.L.T.), Charlottesville.
Abstract
Cardiac sympathetic stimulation activates -adrenergic (-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess -AR effects on NKA function in isolated myocytes. In WT myocytes, 1 eol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5±1.8 to 10.5±1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2±1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9±0.9 versus 16.2±1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).
Key Words: Na pump phospholemman signal transduction ion channels
Introduction
Activation of the sympathetic nervous system and cardiac -adrenergic (-AR) receptors causes cAMP formation and activation of protein kinase A (PKA). In cardiac myocytes, PKA phosphorylates several targets with key roles in the control of excitation-contraction coupling (ECC), including L-type Ca2+ channels, phospholamban (PLB) and troponin-I, as well as other sarcolemmal proteins such as voltage-gated Na and K channels and phospholemman (PLM).
During sympathetic activation, the larger Ca influx via more frequent and larger Ca current must be balanced by enhanced Ca extrusion via the Na/Ca exchange (NCX) that is driven by larger Ca transients. This increases Na influx at each beat, along with more frequent and larger Na current, which increases intracellular [Na] ([Na]i). To limit the rise in [Na]i, the greater Na influx must be compensated for by an enhanced Na extrusion via the Na/K pump (NKA). Indeed, many early studies indicated stimulation of the Na-pump by -AR activation.1eC3 However, there is controversy at present because some recent studies in single myocytes using NKA pump current (IPump) found either stimulation,4eC6 inhibition,7,8 or no change9 in IPump on -AR stimulation.
Controversy extends beyond the direction of -AR effects on NKA function, as to the molecular mechanism involved. NKA subunit can be phosphorylated by PKA only in the presence of detergents or after reconstitution,10,11 whereas in situ the phosphorylation site may be inaccessible to the kinase.12 This raises the question of whether phosphorylation of a NKA regulatory protein could mediate -AR effects.
In heart, such a role could be played by the small transmembrane protein PLM,13,14 long known to be a major cardiac substrate for both PKA and protein kinase C (PKC).15,16 However, the physiological role of PLM is poorly understood. Recent studies show that PLM associates with NKA and decreases its apparent Na and K affinity.13 Furthermore, PLM is one of the 7 members of the FXYD gene family, which also includes the NKA -subunit and CHIF in the kidney, both of which associate with and modulate NKA.17 However, PLM is the only FXYD protein that is known to be phosphorylated. Thus, PLM might affect NKA function in a manner similar to the way PLB affects SERCA, a P-type pump closely related to NKA, ie, inhibition, relieved by phosphorylation.
The aim of this article is to test the hypotheses that (1) -AR stimulation activates NKA by increasing its [Na]i affinity, (2) PLM inhibits NKA activity in a manner analogous to PLB inhibition of SERCA (by decreasing [Na]i affinity), and (3) -AR stimulation of NKA is mediated by PLM phosphorylation. We combined measurements of NKA-mediated [Na]i decline and IPump to determine the effect of -AR activation by isoproterenol (ISO) on NKA function in single myocytes isolated from wild-type (WT) mice and mice in which the PLM gene was targeted (PLM-KO).18 ISO stimulated the pump in the WT mice by reducing the Km for internal Na and phosphorylated PLM at Ser-68. However, ISO had no significant effect in the PLM-KO. Moreover, NKA function in the PLM-KO mice was similar to that in the WT in the presence of ISO.
Materials and Methods
A more detailed Materials and Methods is available in the online data supplement at http://circres.ahajournal.org.
Generation of PLM-KO Mice
PLM-KO mice were generated at the University of Virginia (Charlottesville, Va) as previously described18 except that they are now congenic on a pure C57B/6 background. Heterozygous breeding pairs were used to generate PLM-KO and WT littermates. Mice of 3 to 4 months of age were used, before PLM-KO mice developed hypertrophy.18 All animal protocols were approved by the Animal Care and Use Committee at Loyola University Chicago and University of Virginia.
Myocyte Isolation
Isolation of mouse ventricular myocytes was as previously described.19 Briefly, PLM-KO mice and age-matched WT littermates were anesthetized (with 3% to 5% isoflurane). Hearts were excised quickly and mounted on a Langendorff perfusion apparatus and exposed to 0.8 mg/mL collagenase (type B, Boehringer Mannheim) for 7 to 12 minutes. Ventricular tissue was removed, dispersed, filtered, and myocyte suspensions were rinsed several times. The yield of viable rod-shaped myocytes was 70% to 80%. Cell size was similar in WT and KO myocytes based on membrane capacitance (204±14 versus 215±20 pF, respectively, n=8 each).
Intracellular [Na] Measurements in Intact Myocytes
Isolated myocytes plated on laminin-coated coverslips were loaded with SBFI-AM, and dual excitation fluorescence measurements (at 340 and 380 nm; F340 and F380) were performed as previously described.20,21 F340/F380 was calculated after background subtraction and converted to [Na]i by calibration at the end of each experiment in the presence of 10 eol/L gramicidin and 100 eol/L strophanthidin.21 Generally, [Na]i was measured at 15 to 60 s intervals to minimize indicator photobleaching and cell photodamage. All the measurements were at room temperature.
Na Efflux Through the Na/K-Pump
Na/K pump flux was determined as the rate of pump-mediated [Na]i decline.21 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). NCX is blocked in this Na-free, Ca-free solution, assuring that measured PLM effects on [Na]i decline depend directly on NKA and not secondary to any effect on NCX. Because cell volume does not change with this protocol,22 [Na]i decline reflects Na efflux. The rate of [Na]i decline (eCd[Na]i/dt) was plotted versus [Na]i and fitted with: eCd[Na]i/dt=Vmax/ (1+(Km/[Na]i)nHill).
Simultaneous Na/K Pump Current and [Na]i Measurements
Simultaneous IPump and [Na]i measurements were done as previously described.20 Briefly, thapsigargin pretreated myocytes were whole-cell voltage clamped under conditions that minimized the effect of cell dialysis by the patch pipette on [Na]i while maximizing its control by trans-sarcolemmal Na fluxes. Electrode series resistance (3 to 4 M before patching) was 6 to 12 M during current recording. The standard pipette solution contained (in mmol/L): 10 NaCl, 20 KCl, 100 K-aspartate, 20 TEA-Cl, 10 HEPES, 5 Mg-ATP, 0.7 MgCl2 (1 mmol/L free Mg), 3 BAPTA, 1.15 CaCl2 (100 nM free Ca), 1 SBFI tetraamonium salt, pH=7.2. The external solution contained (in mmol/L): 136 NaCl, 5 NiCl2, 2 BaCl2, 1 MgCl2, 5 HEPES 10 glucose, and 4 KCl (4 mmol/L K solution) or Tris-Cl (0 mmol/L K solution), pH=7.4. IPump was measured at eC20 mV as the outward shift induced by switching from K-free to K-containing external solution. This also corrected for a small variable ISO-activated current attributed to CFTR. We showed previously that the K-sensitive current is equivalent to ouabain-sensitive current.20
Immunoblots and Immunoprecipitations
Isolated myocytes were lysed in ice-cold buffer containing 1% NP-40 and (in mmol/L) 150 NaCl, 10 Tris (pH 7.4), 2 EGTA, 50 NaF, 0.2 NaVO3, and protease inhibitors (Calbiochem). Lysates were then flash frozen and stored at eC80°C. Lysates were also prepared after myocyte treatment with 1 eol/L ISO. Standard Western blots were performed (see online data supplement). NKA alpha subunit and PLM were immunoprecipitated as previously described,13,33 using -isoform-and PLM specific antibodies (see online data supplement).
3H-Ouabain Binding
3H-ouabain binding was measured with a filtration method using isolated myocytes.23,24 Briefly, isolated myocytes were permeabilized (25 e/mL saponin) and incubated at 37°C for 6 hours in solution containing (in mmol/L) 0.1 [3H]ouabain, 0.01 sodium meta-vanadate, 5 MgCl2, 50 Tris-HCl (pH 7.4), 5 Tris-PO4. After incubation myocytes were filtered (Whatman GF/C filter paper) and washed, and filter-associated radioactivity was determined by liquid scintillation counting. Nonspecific 3H-ouabain was determined by using an excess of unlabeled ouabain (1 mmol/L) and [ouabain] dependence was checked to assure that these conditions measure >90% of the number of Na/K-ATPase sites (Bmax).
Sarcolemmal Isolation and ATPase Activity
A sarcolemmal-enriched fraction was prepared as described25 with modifications (see online data supplement). Ouabain-sensitive Na/K-ATPase activity was measured at 37°C in a spectrophotometric enzyme-coupled assay as described.26 Sarcolemma (2 e) was incubated in medium containing (in mmol/L) Tris-HCl 30, KCl 20, MgCl2 3, EDTA 1, Phospho(enol)Pyruvate 15, NADH 1, ATP 3, ±ouabain 10, and 4.6 U of lactic dehydrogenase and 3.3 U of pyruvate kinase at pH 7.2, with [NaCl]=0 to 120 mmol/L. The decline in NADH absorbance at 340 nm is used to calculate ATPase rate sensitive to 10 mmol/L ouabain.
Statistical Analysis
Data are expressed as mean±SEM. Student t test (paired when appropriate) was used for statistical discriminations, with P<0.05 considered significant.
Results
Effect of ISO on the Na/K Pump-Mediated Na Efflux in Myocytes From WT and PLM-KO Mice
The effect of -AR stimulation on NKA function was measured in intact myocytes by measuring the [Na]i-dependence of NKA-mediated Na efflux. Myocytes were Na-loaded by incubation in K-free solution to block NKA (Figure 1A). Then the Na/K-Pump was reactivated by readmission of 4 mmol/L [K]o (and removal of extracellular Na) and the time course of [Na]i decline was measured. The protocol was then repeated in the presence of 1 eol/L ISO (Figure 1A). In the experiment shown in Figure 1A, Na efflux in Na-free solution is mediated by both NKA and other mechanisms (outward leak). To account for the leak, we performed a parallel series of experiments where Na efflux was measured in the presence of 10 mmol/L ouabain, to completely block NKA. [Na]i decline in each case was numerically differentiated and d[Na]i/dt was plotted as a function of [Na]i (Figure 1B). NKA-mediated Na efflux was obtained by subtracting the leak component from the total Na efflux. When 2 consecutive control runs are done, the resulting Na efflux is highly reproducible (see online Figure IS).
Figure 2 shows average data for the effect of ISO on the [Na]i dependence of NKA function in WT and PLM-KO myocytes. Data were fit with a Hill expression to derive the Vmax, Km, and nHill (Table). In WT mice (Figure 2A), ISO stimulated NKA activity, mainly by increasing apparent affinity for [Na]i (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L, n=10, P=0.021). In PLM-KO mice, the Vmax, Km, and nHill under control conditions were similar to the WT in the presence of ISO (Table). Thus -AR stimulation makes the [Na]i-dependence of NKA in myocytes from WT mice similar to that found in the PLM-KO mice. Furthermore, ISO failed to significantly shift Km in the PLM-KO mice (14.6±1.9 versus 13.1±1.5 mmol/L, n=9, P=0.611). ISO did not significantly alter the Vmax or nHill in either WT or PLM-KO myocytes.
The [Na]-dependence of ouabain-sensitive ATPase in sarcolemmal vesicles was also shifted in PLM-KO versus WT ventricle (Km=12.9 versus 16.2 mmol/L; Table). This is similar to the shift based on d[Na]i /dt in myocytes (Km=14.2 versus 18.8 mmol/L; Table) and confirms the PLM-induced inhibition of apparent Na affinity in a more controlled (but less physiological) environment.
Effect of ISO on IPump in Myocytes From WT and PLM-KO Mice
To further test our hypotheses, we determined the effect of -AR stimulation on IPump by simultaneously monitoring IPump and [Na]i under voltage clamp conditions at [Na]i in the range of the Km where differences should be most apparent. In ruptured patch whole-cell configuration, NKA was inhibited for 10 to 12 minutes in a K-free solution (Figure 3). This led to a rise in [Na]i above that in the pipette solution caused by Na entry from the external solution. Then, the pump was abruptly reactivated by restoring K (4 mmol/L) while measuring membrane current (Figure 3A and 3D) and [Na]i (Figure 3B and 3E). IPump decays (Figure 3A and 3D) as [Na]i declines (Figure 3B and 3E).20 The protocol was repeated in the same cell in the presence of 1 eol/L ISO. Figure 3A through 3C shows a representative experiment in myocytes from WT mouse. IPump is larger in the presence of ISO, which results in a faster decrease in [Na]i. The plot of IPump versus [Na]i (Figure 3C) emphasizes that IPump is higher in the presence of ISO for a given [Na]i in a range (9 to 14 mmol/L) comparable to the Km. In contrast, ISO has no effect on IPump in myocytes from PLM-KO mouse (Figure 3D through 3F). Thus, IPump data are consistent with a PLM-dependent stimulation of NKA function by ISO, via an increased affinity for internal Na.
Figure 4A shows pooled data from experiments like that in Figure 3. For an initial [Na]i that is not significantly different on IPump reactivation in any of the 4 cases (Figure 4A; 14.5 mmol/L) mean peak IPump is significantly increased by ISO in WT but not PLM-KO myocytes. This further confirms that ISO stimulation of NKA depends on the presence of PLM.
Phosphorylation of Phospholemman on -AR Stimulation and PLM-NKA Association
Figure 5A shows that PLM was indeed undetectable in PLM-KO myocytes, but there was also downregulation of NKA expression (assessed by an antibody that detects all 3 isoforms of NKA equally). This might explain why IPump in ISO-stimulated WT myocytes is larger than in PLM-KO cells (Figure 4B). Immunoblots with phospho-specific antibodies (Figure 5B) show an increase in PLM phosphorylation at Ser-68 (PKA site), but not at Ser-63 (the PKC site) in myocytes exposed to ISO for 15 minutes. Pooled data from Western blots indicated a 20% reduction in NKA expression (Figure 5C), and this was very similar to the 25% reduction in Bmax detected in 3H-ouabain binding assays (Figure 5D). Thus, in mice without tonic PLM-dependent NKA inhibition (which would increase NKA activity), there appears to be an adaptation to reduce the number of Na-pumps. This smaller number of Na-pumps in the PLM-KO and their inability to be regulated by -AR activation would limit the ability of cells to increase Na extrusion in response to sympathetic activation.
Figure 5E shows that PLM and NKA 1-subunit coimmunoprecipitate. Thus, there is both a physical association and functional interaction between PLM and NKA in mouse ventricular myocytes.
Effect of ISO on Resting [Na]i in Myocytes From WT and PLM-KO Mice
Figure 6 shows resting [Na]i in WT and PLM-KO myocytes and the effect of 1 eol/L ISO. Resting [Na]i is determined by a balance of Na efflux via NKA and Na influx via several mechanisms (NCX, Na/H exchange, Na channels).27 Resting [Na]i was comparable in myocytes from the WT and PLM-KO mice under basal conditions (12.5±1.8 versus 12.0±1.5 mmol/L). This is consistent with the notion that the increased Na affinity of NKA in PLM-KO mice might be offset by a lower number of NKA molecules, resulting in relatively normal [Na]i and NKA rate at that [Na]i.
In resting WT mice, ISO stimulation significantly reduced [Na]i to 10.5±1.4 mmol/L (Figure 6). In 4 of 5 cells investigated, this was reversible on ISO washout (not shown). On the other hand, ISO had no significant effect on resting [Na]i in PLM-KO mice. This extends the observation from direct measurements of IPump and d[Na]i/dt, that [Na]i regulation in resting ventricular myocytes by -AR is mediated by PLM.
Discussion
The present study demonstrates that PLM regulates the Na/K pump in a manner similar to the way PLB modulates SERCA, ie, it inhibits the pump by decreasing the affinity for intracellular Na (Km=14.2±1.5 mmol/L in PLM-KO mouse versus 18.8±1.4 mmol/L in WT) and the inhibition is relieved on PLM phosphorylation (Km=13.6±1.5 mmol/L in WT mice with ISO). Furthermore, the lack of NKA stimulation by ISO in PLM-KO mouse indicates that -AR effects on NKA are mediated primarily by PLM, rather than direct phosphorylation of NKA.
-AR Stimulation and NKA Activity
Historically, numerous studies indicated that -AR stimulation enhances NKA activity and lowers [Na]i in heart (eg, see references in Glitsch et al2). However, there is recent controversy. Most studies at the single cell level, using either IPump (voltage-clamp) or [Na]i measurements indicate that NKA function is increased by -AR stimulation (present study1,4eC6;). However, some voltage-clamp studies have reported either no effect of -AR on IPump in rat9 or even an inhibition of guinea-pig IPump that was [Ca]i-dependent (decreased IPump when pipette [Ca] was <150 nM, but increased IPump at high pipette [Ca]).7,8 It has also been reported that -AR stimulation of IPump only affects the 1 isoform of NKA (the major form in most hearts), but not 2.6,28
Both IPump and [Na]i measurements have advantages and disadvantages in assessing the effect of -AR stimulation on NKA. [Na]i measurements, especially using fluorescent indicators, are less invasive as they are done in intact cells without altering the cellular environment. However, the rate of [Na]i change (d[Na]i/dt) is an indirect measure of Na-pump flux that could be affected by changes in cellular Na buffering or cell volume (although neither is expected to change during -AR stimulation). In IPump measurements, cell dialysis by the patch pipette can partly control the intracellular environment, but also alter intracellular composition (eg, of important messengers) and can be subject to contaminating ionic currents.
Here, we found evidence for stimulation of NKA function by -AR in 3 contexts: (1) in nondialyzed myocytes (as the [Na]i-dependence of pump-mediated [Na]i decline on abrupt NKA re-activation), (2) in cells under voltage clamp (simultaneous IPump and [Na]i decline measurements), and (3) in intact quiescent myocytes at physiological levels of [Na] and [K]. Our data in intact WT mouse ventricular myocytes indicate that -AR activation stimulates Na-pump activity by increasing the NKA affinity for internal Na without significantly altering maximal NKA rate (Figure 2A and Table). This conclusion is supported by the IPump and [Na]i decline results in voltage-clamped cells, as they show an increased IPump for a given [Na]i near the Km in the presence of ISO (Figures 3 and 4). -AR-induced NKA stimulation is also consistent with the decline in resting [Na]i on ISO stimulation in otherwise unperturbed myocytes (Figure 6).
PLM Modulation of NKA Function
PLM, a 72eCamino acid sarcolemmal protein expressed highly in heart and brain, is a member of the FXYD protein family (named for a conserved Pro-Phe-X-Tyr-Asp motif).29 FXYD proteins have a single membrane span and include the NKA -subunit (FXYD-2), the regulator of renal NKA (FXYD-4, or CHIF), and the PLM-like shark rectal gland protein (PLMS). PLM, FXYD-2, -4, -7, and PLMS all coimmunoprecipitate with NKA subunits and modulate NKA function,6,13,30eC33 but how PLM regulates cardiac NKA is unclear. Crambert et al13 showed that, when transfected into Xenopus oocytes, PLM associates specifically (stoichiometrically) and stably with rat NKA 1 and 2 subunits and that PLM inhibits NKA by reducing its apparent affinity for [Na]i (Km increased from 9.3 to 16.5 mmol/L) and [K]o (Km increased from 0.49 to 0.67 mmol/L).
Initial data in PLM-KO mice showed greater reduction of maximal Na/K-ATPase activity in a sarcolemma-enriched fraction than for NKA expression in homogenates (20%),18 implying that PLM expression might activate NKA. Here we found a similar degree of downregulation of NKA subunit in PLM-KO based on myocyte 3H-ouabain binding (25%), myocyte Western blot (20%), and membrane fraction Na/K-ATPase (17%, nonsignificant), but no decrease in Vmax for d[Na]i/dt in PLM-KO versus WT myocytes (Table). Possibly the stronger depression of plasmalemmal Na/K-ATPase Vmax reported in the prior study18 was complicated by membranes from nonmyocytes, differential sarcolemmal enrichment, or some other assay factor.
Our results here with PLM-KO mice indicate that PLM depresses NKA activity, mainly via reduced [Na]i affinity (by 4 mmol/L), but an effect of PLM on Vmax cannot be ruled out. That is, PLM-KO myocytes had reduced NKA protein expression (20% to 25%) with a tendency toward higher Vmax in d[Na]i/dt (10%, though not significant). Notably, ISO also tended to increase Vmax of d[Na]i/dt by 11%, but this was also true in PLM-KO (Table). Small apparent changes in Vmax cannot be interpreted clearly here, because they could be caused by numerous other effects (eg, increased K affinity of NKA13 or fraction of total NKA molecules which are on the sarcolemma and functional). However, this does leave open the possibility that PKA could alter Vmax (which we find significant in rabbit ventricular myocytes, not shown) and that may or may not be regulated by PLM phosphorylation. Thus further study will be required to understand whether PLM alters NKA Vmax and whether PLM phosphorylation alters that regulation. However, the effect of PLM on the Na affinity of NKA and in mediating the change in Km(Nai) induced by ISO that we show here is compelling. This functional interaction between PLM and NKA seems to be based on physical association, as indicated by coimmunoprecipitation data.6,13,33
Despite NKA inhibition by PLM, resting [Na]i is not higher in myocytes from WT versus PLM-KO mice. This might be attributable in part to the reduced NKA expression in PLM-KO mice (Figure 5). The similar resting [Na]i in WT and PLM-KO myocytes could also be partly attributable to increased Na influx in PLM-KO mice. Using the initial rate of [Na]i rise on NKA blockade (as in Figure 1) the mean apparent Na influx rate was slightly higher in PLM-KO versus WT myocytes (3.1±0.5 versus 2.7±0.4 mmol/L/min at 12 mmol/L [Na]i, n=14), but the difference was not significant.
PLM Phosphorylation
PLM is unique in the FXYD family in having multiple cytosolic phosphorylation sites and is a major cardiac target for -AR mediated phosphorylation. Quantitatively, PLM phosphorylation by PKA is comparable to that of troponin I and PLB.15,34 PLM is phosphorylated by ISO under the conditions used here (Figure 5). In PLM-KO mice ISO did not significantly alter either Vmax and Km of the Na/K pump or resting [Na]i. This is most consistent with the -AR-dependent NKA stimulation in WT mice being mediated by PLM phosphorylation. After -AR stimulation NKA function in WT mice is almost identical to that in PLM-KO mice without ISO. Thus, PLM inhibits NKA (by decreasing its affinity for [Na]i), and this inhibition is relieved on PLM phosphorylation. This is highly analogous to the way PLB modulates SERCA activity.
Interestingly, the association between PLM and NKA, as determined by coIP experiments, appears to be unaffected by PKA phosphorylation.14,33 Thus phosphorylation may change the PLM-NKA interaction but does not necessarily result in a complete dissociation. In the analogous PLB-SERCA system it was long thought that PLB phosphorylation caused it to dissociate from SERCA, but more recent results show that PLB remains bound to SERCA even after phosphorylation and abolition of SERCA inhibition.35
Physiological Context of -AR Activation of NKA
During -AR stimulation in the sympathetic fight or flight response, Na influx into cardiac myocytes is greatly increased. This is attributable to more frequent Na current (which may also be larger caused by PKA36) and more Na entry via NCX, that is both because of higher frequency and because larger Ca transients drive greater Ca extrusion coupled Na influx at each beat. Looked at another way, the higher Ca current on -AR stimulation increases Ca influx, and Ca extrusion (coupled to Na influx via NCX) must also be increased to attain Ca flux balance. Indeed, Na entry via NCX is a major fraction of Na influx during the cardiac cycle.27 Enhancement of NKA activity may thus be an integral part of the sympathetic response of the heart to enhance Na extrusion to better keep up with the higher level of Na influx. This may limit the rise in [Na]i that occurs during the combined inotropic and chronotropic effects on the heart. Our data show that NKA stimulation by -AR activation results in reduced [Na]i in resting myocytes from WT but not PLM-KO mice (Figure 6). The inability of NKA to be stimulated by -AR in the PLM-KO mouse could lead to excessive elevation of [Na]i during sympathetic activation, which might have both the benefits and the risks associated with NKA inhibition by glycosides (inotropy, but enhanced arrhythmogenesis). This process might be further complicated by possible direct effects of PLM on NCX.37
In summary, we have shown that (1) -AR stimulation activates NKA in mouse cardiac myocytes by increasing its affinity for internal Na, with no significant effect on the maximum pump rate, (2) PLM inhibits NKA activity by decreasing its [Na]i affinity, and (3) -AR stimulation of NKA is mediated by PLM phosphorylation. Thus, our data indicate that PLM modulates the NKA function in a manner similar to the way PLB affects SERCA.
Acknowledgments
This work was supported by grants from the National Institutes of Health (HL-30077, HL-64724; D.M.B.), the Cardiovascular Research Center at University of Virginia (A.L.T.), and fellowships from the American Heart Association (S.D., J.B.). The authors thank Brian French and Jaime O’Brien for myocyte preparation.
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the University of Virginia (L.J., A.L.T.), Charlottesville.
Abstract
Cardiac sympathetic stimulation activates -adrenergic (-AR) receptors and protein kinase A (PKA) phosphorylation of proteins involved in myocyte Ca regulation. The Na/K-ATPase (NKA) is essential in regulating intracellular [Na] ([Na]i), which in turn affects [Ca]i via Na/Ca exchange. However, how PKA modifies NKA function is unknown. Phospholemman (PLM), a member of the FXYD family of proteins that interact with NKA in various tissues, is a major PKA substrate in heart. Here we tested the hypothesis that PLM phosphorylation is responsible for the PKA effects on cardiac NKA function using wild-type (WT) and PLM knockout (PLM-KO) mice. We measured NKA-mediated [Na]i decline and current (IPump) to assess -AR effects on NKA function in isolated myocytes. In WT myocytes, 1 eol/L isoproterenol (ISO) increased PLM phosphorylation and stimulated NKA activity mainly by increasing its affinity for internal Na (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L), with no significant effect on the maximum pump rate. This led to a significant decrease in resting [Na]i (from 12.5±1.8 to 10.5±1.4 mmol/L). In PLM-KO mice under control conditions Km (14.2±1.5 mmol/L) was lower than in WT, but comparable to that for WT in the presence of ISO. Furthermore, ISO had no significant effect on NKA function in PLM-KO mice. ATPase activity in sarcolemmal vesicles also showed a lower Km(Na) in PLM-KO versus WT (12.9±0.9 versus 16.2±1.5). Thus, PLM inhibits NKA activity by decreasing its [Na]i affinity, and this inhibitory effect is relieved by PKA activation. We conclude that PLM modulates the NKA function in a manner similar to the way phospholamban affects the related SR Ca-ATPase (inhibition of transport substrate affinity, that is relieved by phosphorylation).
Key Words: Na pump phospholemman signal transduction ion channels
Introduction
Activation of the sympathetic nervous system and cardiac -adrenergic (-AR) receptors causes cAMP formation and activation of protein kinase A (PKA). In cardiac myocytes, PKA phosphorylates several targets with key roles in the control of excitation-contraction coupling (ECC), including L-type Ca2+ channels, phospholamban (PLB) and troponin-I, as well as other sarcolemmal proteins such as voltage-gated Na and K channels and phospholemman (PLM).
During sympathetic activation, the larger Ca influx via more frequent and larger Ca current must be balanced by enhanced Ca extrusion via the Na/Ca exchange (NCX) that is driven by larger Ca transients. This increases Na influx at each beat, along with more frequent and larger Na current, which increases intracellular [Na] ([Na]i). To limit the rise in [Na]i, the greater Na influx must be compensated for by an enhanced Na extrusion via the Na/K pump (NKA). Indeed, many early studies indicated stimulation of the Na-pump by -AR activation.1eC3 However, there is controversy at present because some recent studies in single myocytes using NKA pump current (IPump) found either stimulation,4eC6 inhibition,7,8 or no change9 in IPump on -AR stimulation.
Controversy extends beyond the direction of -AR effects on NKA function, as to the molecular mechanism involved. NKA subunit can be phosphorylated by PKA only in the presence of detergents or after reconstitution,10,11 whereas in situ the phosphorylation site may be inaccessible to the kinase.12 This raises the question of whether phosphorylation of a NKA regulatory protein could mediate -AR effects.
In heart, such a role could be played by the small transmembrane protein PLM,13,14 long known to be a major cardiac substrate for both PKA and protein kinase C (PKC).15,16 However, the physiological role of PLM is poorly understood. Recent studies show that PLM associates with NKA and decreases its apparent Na and K affinity.13 Furthermore, PLM is one of the 7 members of the FXYD gene family, which also includes the NKA -subunit and CHIF in the kidney, both of which associate with and modulate NKA.17 However, PLM is the only FXYD protein that is known to be phosphorylated. Thus, PLM might affect NKA function in a manner similar to the way PLB affects SERCA, a P-type pump closely related to NKA, ie, inhibition, relieved by phosphorylation.
The aim of this article is to test the hypotheses that (1) -AR stimulation activates NKA by increasing its [Na]i affinity, (2) PLM inhibits NKA activity in a manner analogous to PLB inhibition of SERCA (by decreasing [Na]i affinity), and (3) -AR stimulation of NKA is mediated by PLM phosphorylation. We combined measurements of NKA-mediated [Na]i decline and IPump to determine the effect of -AR activation by isoproterenol (ISO) on NKA function in single myocytes isolated from wild-type (WT) mice and mice in which the PLM gene was targeted (PLM-KO).18 ISO stimulated the pump in the WT mice by reducing the Km for internal Na and phosphorylated PLM at Ser-68. However, ISO had no significant effect in the PLM-KO. Moreover, NKA function in the PLM-KO mice was similar to that in the WT in the presence of ISO.
Materials and Methods
A more detailed Materials and Methods is available in the online data supplement at http://circres.ahajournal.org.
Generation of PLM-KO Mice
PLM-KO mice were generated at the University of Virginia (Charlottesville, Va) as previously described18 except that they are now congenic on a pure C57B/6 background. Heterozygous breeding pairs were used to generate PLM-KO and WT littermates. Mice of 3 to 4 months of age were used, before PLM-KO mice developed hypertrophy.18 All animal protocols were approved by the Animal Care and Use Committee at Loyola University Chicago and University of Virginia.
Myocyte Isolation
Isolation of mouse ventricular myocytes was as previously described.19 Briefly, PLM-KO mice and age-matched WT littermates were anesthetized (with 3% to 5% isoflurane). Hearts were excised quickly and mounted on a Langendorff perfusion apparatus and exposed to 0.8 mg/mL collagenase (type B, Boehringer Mannheim) for 7 to 12 minutes. Ventricular tissue was removed, dispersed, filtered, and myocyte suspensions were rinsed several times. The yield of viable rod-shaped myocytes was 70% to 80%. Cell size was similar in WT and KO myocytes based on membrane capacitance (204±14 versus 215±20 pF, respectively, n=8 each).
Intracellular [Na] Measurements in Intact Myocytes
Isolated myocytes plated on laminin-coated coverslips were loaded with SBFI-AM, and dual excitation fluorescence measurements (at 340 and 380 nm; F340 and F380) were performed as previously described.20,21 F340/F380 was calculated after background subtraction and converted to [Na]i by calibration at the end of each experiment in the presence of 10 eol/L gramicidin and 100 eol/L strophanthidin.21 Generally, [Na]i was measured at 15 to 60 s intervals to minimize indicator photobleaching and cell photodamage. All the measurements were at room temperature.
Na Efflux Through the Na/K-Pump
Na/K pump flux was determined as the rate of pump-mediated [Na]i decline.21 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). NCX is blocked in this Na-free, Ca-free solution, assuring that measured PLM effects on [Na]i decline depend directly on NKA and not secondary to any effect on NCX. Because cell volume does not change with this protocol,22 [Na]i decline reflects Na efflux. The rate of [Na]i decline (eCd[Na]i/dt) was plotted versus [Na]i and fitted with: eCd[Na]i/dt=Vmax/ (1+(Km/[Na]i)nHill).
Simultaneous Na/K Pump Current and [Na]i Measurements
Simultaneous IPump and [Na]i measurements were done as previously described.20 Briefly, thapsigargin pretreated myocytes were whole-cell voltage clamped under conditions that minimized the effect of cell dialysis by the patch pipette on [Na]i while maximizing its control by trans-sarcolemmal Na fluxes. Electrode series resistance (3 to 4 M before patching) was 6 to 12 M during current recording. The standard pipette solution contained (in mmol/L): 10 NaCl, 20 KCl, 100 K-aspartate, 20 TEA-Cl, 10 HEPES, 5 Mg-ATP, 0.7 MgCl2 (1 mmol/L free Mg), 3 BAPTA, 1.15 CaCl2 (100 nM free Ca), 1 SBFI tetraamonium salt, pH=7.2. The external solution contained (in mmol/L): 136 NaCl, 5 NiCl2, 2 BaCl2, 1 MgCl2, 5 HEPES 10 glucose, and 4 KCl (4 mmol/L K solution) or Tris-Cl (0 mmol/L K solution), pH=7.4. IPump was measured at eC20 mV as the outward shift induced by switching from K-free to K-containing external solution. This also corrected for a small variable ISO-activated current attributed to CFTR. We showed previously that the K-sensitive current is equivalent to ouabain-sensitive current.20
Immunoblots and Immunoprecipitations
Isolated myocytes were lysed in ice-cold buffer containing 1% NP-40 and (in mmol/L) 150 NaCl, 10 Tris (pH 7.4), 2 EGTA, 50 NaF, 0.2 NaVO3, and protease inhibitors (Calbiochem). Lysates were then flash frozen and stored at eC80°C. Lysates were also prepared after myocyte treatment with 1 eol/L ISO. Standard Western blots were performed (see online data supplement). NKA alpha subunit and PLM were immunoprecipitated as previously described,13,33 using -isoform-and PLM specific antibodies (see online data supplement).
3H-Ouabain Binding
3H-ouabain binding was measured with a filtration method using isolated myocytes.23,24 Briefly, isolated myocytes were permeabilized (25 e/mL saponin) and incubated at 37°C for 6 hours in solution containing (in mmol/L) 0.1 [3H]ouabain, 0.01 sodium meta-vanadate, 5 MgCl2, 50 Tris-HCl (pH 7.4), 5 Tris-PO4. After incubation myocytes were filtered (Whatman GF/C filter paper) and washed, and filter-associated radioactivity was determined by liquid scintillation counting. Nonspecific 3H-ouabain was determined by using an excess of unlabeled ouabain (1 mmol/L) and [ouabain] dependence was checked to assure that these conditions measure >90% of the number of Na/K-ATPase sites (Bmax).
Sarcolemmal Isolation and ATPase Activity
A sarcolemmal-enriched fraction was prepared as described25 with modifications (see online data supplement). Ouabain-sensitive Na/K-ATPase activity was measured at 37°C in a spectrophotometric enzyme-coupled assay as described.26 Sarcolemma (2 e) was incubated in medium containing (in mmol/L) Tris-HCl 30, KCl 20, MgCl2 3, EDTA 1, Phospho(enol)Pyruvate 15, NADH 1, ATP 3, ±ouabain 10, and 4.6 U of lactic dehydrogenase and 3.3 U of pyruvate kinase at pH 7.2, with [NaCl]=0 to 120 mmol/L. The decline in NADH absorbance at 340 nm is used to calculate ATPase rate sensitive to 10 mmol/L ouabain.
Statistical Analysis
Data are expressed as mean±SEM. Student t test (paired when appropriate) was used for statistical discriminations, with P<0.05 considered significant.
Results
Effect of ISO on the Na/K Pump-Mediated Na Efflux in Myocytes From WT and PLM-KO Mice
The effect of -AR stimulation on NKA function was measured in intact myocytes by measuring the [Na]i-dependence of NKA-mediated Na efflux. Myocytes were Na-loaded by incubation in K-free solution to block NKA (Figure 1A). Then the Na/K-Pump was reactivated by readmission of 4 mmol/L [K]o (and removal of extracellular Na) and the time course of [Na]i decline was measured. The protocol was then repeated in the presence of 1 eol/L ISO (Figure 1A). In the experiment shown in Figure 1A, Na efflux in Na-free solution is mediated by both NKA and other mechanisms (outward leak). To account for the leak, we performed a parallel series of experiments where Na efflux was measured in the presence of 10 mmol/L ouabain, to completely block NKA. [Na]i decline in each case was numerically differentiated and d[Na]i/dt was plotted as a function of [Na]i (Figure 1B). NKA-mediated Na efflux was obtained by subtracting the leak component from the total Na efflux. When 2 consecutive control runs are done, the resulting Na efflux is highly reproducible (see online Figure IS).
Figure 2 shows average data for the effect of ISO on the [Na]i dependence of NKA function in WT and PLM-KO myocytes. Data were fit with a Hill expression to derive the Vmax, Km, and nHill (Table). In WT mice (Figure 2A), ISO stimulated NKA activity, mainly by increasing apparent affinity for [Na]i (Km decreased from 18.8±1.4 to 13.6±1.5 mmol/L, n=10, P=0.021). In PLM-KO mice, the Vmax, Km, and nHill under control conditions were similar to the WT in the presence of ISO (Table). Thus -AR stimulation makes the [Na]i-dependence of NKA in myocytes from WT mice similar to that found in the PLM-KO mice. Furthermore, ISO failed to significantly shift Km in the PLM-KO mice (14.6±1.9 versus 13.1±1.5 mmol/L, n=9, P=0.611). ISO did not significantly alter the Vmax or nHill in either WT or PLM-KO myocytes.
The [Na]-dependence of ouabain-sensitive ATPase in sarcolemmal vesicles was also shifted in PLM-KO versus WT ventricle (Km=12.9 versus 16.2 mmol/L; Table). This is similar to the shift based on d[Na]i /dt in myocytes (Km=14.2 versus 18.8 mmol/L; Table) and confirms the PLM-induced inhibition of apparent Na affinity in a more controlled (but less physiological) environment.
Effect of ISO on IPump in Myocytes From WT and PLM-KO Mice
To further test our hypotheses, we determined the effect of -AR stimulation on IPump by simultaneously monitoring IPump and [Na]i under voltage clamp conditions at [Na]i in the range of the Km where differences should be most apparent. In ruptured patch whole-cell configuration, NKA was inhibited for 10 to 12 minutes in a K-free solution (Figure 3). This led to a rise in [Na]i above that in the pipette solution caused by Na entry from the external solution. Then, the pump was abruptly reactivated by restoring K (4 mmol/L) while measuring membrane current (Figure 3A and 3D) and [Na]i (Figure 3B and 3E). IPump decays (Figure 3A and 3D) as [Na]i declines (Figure 3B and 3E).20 The protocol was repeated in the same cell in the presence of 1 eol/L ISO. Figure 3A through 3C shows a representative experiment in myocytes from WT mouse. IPump is larger in the presence of ISO, which results in a faster decrease in [Na]i. The plot of IPump versus [Na]i (Figure 3C) emphasizes that IPump is higher in the presence of ISO for a given [Na]i in a range (9 to 14 mmol/L) comparable to the Km. In contrast, ISO has no effect on IPump in myocytes from PLM-KO mouse (Figure 3D through 3F). Thus, IPump data are consistent with a PLM-dependent stimulation of NKA function by ISO, via an increased affinity for internal Na.
Figure 4A shows pooled data from experiments like that in Figure 3. For an initial [Na]i that is not significantly different on IPump reactivation in any of the 4 cases (Figure 4A; 14.5 mmol/L) mean peak IPump is significantly increased by ISO in WT but not PLM-KO myocytes. This further confirms that ISO stimulation of NKA depends on the presence of PLM.
Phosphorylation of Phospholemman on -AR Stimulation and PLM-NKA Association
Figure 5A shows that PLM was indeed undetectable in PLM-KO myocytes, but there was also downregulation of NKA expression (assessed by an antibody that detects all 3 isoforms of NKA equally). This might explain why IPump in ISO-stimulated WT myocytes is larger than in PLM-KO cells (Figure 4B). Immunoblots with phospho-specific antibodies (Figure 5B) show an increase in PLM phosphorylation at Ser-68 (PKA site), but not at Ser-63 (the PKC site) in myocytes exposed to ISO for 15 minutes. Pooled data from Western blots indicated a 20% reduction in NKA expression (Figure 5C), and this was very similar to the 25% reduction in Bmax detected in 3H-ouabain binding assays (Figure 5D). Thus, in mice without tonic PLM-dependent NKA inhibition (which would increase NKA activity), there appears to be an adaptation to reduce the number of Na-pumps. This smaller number of Na-pumps in the PLM-KO and their inability to be regulated by -AR activation would limit the ability of cells to increase Na extrusion in response to sympathetic activation.
Figure 5E shows that PLM and NKA 1-subunit coimmunoprecipitate. Thus, there is both a physical association and functional interaction between PLM and NKA in mouse ventricular myocytes.
Effect of ISO on Resting [Na]i in Myocytes From WT and PLM-KO Mice
Figure 6 shows resting [Na]i in WT and PLM-KO myocytes and the effect of 1 eol/L ISO. Resting [Na]i is determined by a balance of Na efflux via NKA and Na influx via several mechanisms (NCX, Na/H exchange, Na channels).27 Resting [Na]i was comparable in myocytes from the WT and PLM-KO mice under basal conditions (12.5±1.8 versus 12.0±1.5 mmol/L). This is consistent with the notion that the increased Na affinity of NKA in PLM-KO mice might be offset by a lower number of NKA molecules, resulting in relatively normal [Na]i and NKA rate at that [Na]i.
In resting WT mice, ISO stimulation significantly reduced [Na]i to 10.5±1.4 mmol/L (Figure 6). In 4 of 5 cells investigated, this was reversible on ISO washout (not shown). On the other hand, ISO had no significant effect on resting [Na]i in PLM-KO mice. This extends the observation from direct measurements of IPump and d[Na]i/dt, that [Na]i regulation in resting ventricular myocytes by -AR is mediated by PLM.
Discussion
The present study demonstrates that PLM regulates the Na/K pump in a manner similar to the way PLB modulates SERCA, ie, it inhibits the pump by decreasing the affinity for intracellular Na (Km=14.2±1.5 mmol/L in PLM-KO mouse versus 18.8±1.4 mmol/L in WT) and the inhibition is relieved on PLM phosphorylation (Km=13.6±1.5 mmol/L in WT mice with ISO). Furthermore, the lack of NKA stimulation by ISO in PLM-KO mouse indicates that -AR effects on NKA are mediated primarily by PLM, rather than direct phosphorylation of NKA.
-AR Stimulation and NKA Activity
Historically, numerous studies indicated that -AR stimulation enhances NKA activity and lowers [Na]i in heart (eg, see references in Glitsch et al2). However, there is recent controversy. Most studies at the single cell level, using either IPump (voltage-clamp) or [Na]i measurements indicate that NKA function is increased by -AR stimulation (present study1,4eC6;). However, some voltage-clamp studies have reported either no effect of -AR on IPump in rat9 or even an inhibition of guinea-pig IPump that was [Ca]i-dependent (decreased IPump when pipette [Ca] was <150 nM, but increased IPump at high pipette [Ca]).7,8 It has also been reported that -AR stimulation of IPump only affects the 1 isoform of NKA (the major form in most hearts), but not 2.6,28
Both IPump and [Na]i measurements have advantages and disadvantages in assessing the effect of -AR stimulation on NKA. [Na]i measurements, especially using fluorescent indicators, are less invasive as they are done in intact cells without altering the cellular environment. However, the rate of [Na]i change (d[Na]i/dt) is an indirect measure of Na-pump flux that could be affected by changes in cellular Na buffering or cell volume (although neither is expected to change during -AR stimulation). In IPump measurements, cell dialysis by the patch pipette can partly control the intracellular environment, but also alter intracellular composition (eg, of important messengers) and can be subject to contaminating ionic currents.
Here, we found evidence for stimulation of NKA function by -AR in 3 contexts: (1) in nondialyzed myocytes (as the [Na]i-dependence of pump-mediated [Na]i decline on abrupt NKA re-activation), (2) in cells under voltage clamp (simultaneous IPump and [Na]i decline measurements), and (3) in intact quiescent myocytes at physiological levels of [Na] and [K]. Our data in intact WT mouse ventricular myocytes indicate that -AR activation stimulates Na-pump activity by increasing the NKA affinity for internal Na without significantly altering maximal NKA rate (Figure 2A and Table). This conclusion is supported by the IPump and [Na]i decline results in voltage-clamped cells, as they show an increased IPump for a given [Na]i near the Km in the presence of ISO (Figures 3 and 4). -AR-induced NKA stimulation is also consistent with the decline in resting [Na]i on ISO stimulation in otherwise unperturbed myocytes (Figure 6).
PLM Modulation of NKA Function
PLM, a 72eCamino acid sarcolemmal protein expressed highly in heart and brain, is a member of the FXYD protein family (named for a conserved Pro-Phe-X-Tyr-Asp motif).29 FXYD proteins have a single membrane span and include the NKA -subunit (FXYD-2), the regulator of renal NKA (FXYD-4, or CHIF), and the PLM-like shark rectal gland protein (PLMS). PLM, FXYD-2, -4, -7, and PLMS all coimmunoprecipitate with NKA subunits and modulate NKA function,6,13,30eC33 but how PLM regulates cardiac NKA is unclear. Crambert et al13 showed that, when transfected into Xenopus oocytes, PLM associates specifically (stoichiometrically) and stably with rat NKA 1 and 2 subunits and that PLM inhibits NKA by reducing its apparent affinity for [Na]i (Km increased from 9.3 to 16.5 mmol/L) and [K]o (Km increased from 0.49 to 0.67 mmol/L).
Initial data in PLM-KO mice showed greater reduction of maximal Na/K-ATPase activity in a sarcolemma-enriched fraction than for NKA expression in homogenates (20%),18 implying that PLM expression might activate NKA. Here we found a similar degree of downregulation of NKA subunit in PLM-KO based on myocyte 3H-ouabain binding (25%), myocyte Western blot (20%), and membrane fraction Na/K-ATPase (17%, nonsignificant), but no decrease in Vmax for d[Na]i/dt in PLM-KO versus WT myocytes (Table). Possibly the stronger depression of plasmalemmal Na/K-ATPase Vmax reported in the prior study18 was complicated by membranes from nonmyocytes, differential sarcolemmal enrichment, or some other assay factor.
Our results here with PLM-KO mice indicate that PLM depresses NKA activity, mainly via reduced [Na]i affinity (by 4 mmol/L), but an effect of PLM on Vmax cannot be ruled out. That is, PLM-KO myocytes had reduced NKA protein expression (20% to 25%) with a tendency toward higher Vmax in d[Na]i/dt (10%, though not significant). Notably, ISO also tended to increase Vmax of d[Na]i/dt by 11%, but this was also true in PLM-KO (Table). Small apparent changes in Vmax cannot be interpreted clearly here, because they could be caused by numerous other effects (eg, increased K affinity of NKA13 or fraction of total NKA molecules which are on the sarcolemma and functional). However, this does leave open the possibility that PKA could alter Vmax (which we find significant in rabbit ventricular myocytes, not shown) and that may or may not be regulated by PLM phosphorylation. Thus further study will be required to understand whether PLM alters NKA Vmax and whether PLM phosphorylation alters that regulation. However, the effect of PLM on the Na affinity of NKA and in mediating the change in Km(Nai) induced by ISO that we show here is compelling. This functional interaction between PLM and NKA seems to be based on physical association, as indicated by coimmunoprecipitation data.6,13,33
Despite NKA inhibition by PLM, resting [Na]i is not higher in myocytes from WT versus PLM-KO mice. This might be attributable in part to the reduced NKA expression in PLM-KO mice (Figure 5). The similar resting [Na]i in WT and PLM-KO myocytes could also be partly attributable to increased Na influx in PLM-KO mice. Using the initial rate of [Na]i rise on NKA blockade (as in Figure 1) the mean apparent Na influx rate was slightly higher in PLM-KO versus WT myocytes (3.1±0.5 versus 2.7±0.4 mmol/L/min at 12 mmol/L [Na]i, n=14), but the difference was not significant.
PLM Phosphorylation
PLM is unique in the FXYD family in having multiple cytosolic phosphorylation sites and is a major cardiac target for -AR mediated phosphorylation. Quantitatively, PLM phosphorylation by PKA is comparable to that of troponin I and PLB.15,34 PLM is phosphorylated by ISO under the conditions used here (Figure 5). In PLM-KO mice ISO did not significantly alter either Vmax and Km of the Na/K pump or resting [Na]i. This is most consistent with the -AR-dependent NKA stimulation in WT mice being mediated by PLM phosphorylation. After -AR stimulation NKA function in WT mice is almost identical to that in PLM-KO mice without ISO. Thus, PLM inhibits NKA (by decreasing its affinity for [Na]i), and this inhibition is relieved on PLM phosphorylation. This is highly analogous to the way PLB modulates SERCA activity.
Interestingly, the association between PLM and NKA, as determined by coIP experiments, appears to be unaffected by PKA phosphorylation.14,33 Thus phosphorylation may change the PLM-NKA interaction but does not necessarily result in a complete dissociation. In the analogous PLB-SERCA system it was long thought that PLB phosphorylation caused it to dissociate from SERCA, but more recent results show that PLB remains bound to SERCA even after phosphorylation and abolition of SERCA inhibition.35
Physiological Context of -AR Activation of NKA
During -AR stimulation in the sympathetic fight or flight response, Na influx into cardiac myocytes is greatly increased. This is attributable to more frequent Na current (which may also be larger caused by PKA36) and more Na entry via NCX, that is both because of higher frequency and because larger Ca transients drive greater Ca extrusion coupled Na influx at each beat. Looked at another way, the higher Ca current on -AR stimulation increases Ca influx, and Ca extrusion (coupled to Na influx via NCX) must also be increased to attain Ca flux balance. Indeed, Na entry via NCX is a major fraction of Na influx during the cardiac cycle.27 Enhancement of NKA activity may thus be an integral part of the sympathetic response of the heart to enhance Na extrusion to better keep up with the higher level of Na influx. This may limit the rise in [Na]i that occurs during the combined inotropic and chronotropic effects on the heart. Our data show that NKA stimulation by -AR activation results in reduced [Na]i in resting myocytes from WT but not PLM-KO mice (Figure 6). The inability of NKA to be stimulated by -AR in the PLM-KO mouse could lead to excessive elevation of [Na]i during sympathetic activation, which might have both the benefits and the risks associated with NKA inhibition by glycosides (inotropy, but enhanced arrhythmogenesis). This process might be further complicated by possible direct effects of PLM on NCX.37
In summary, we have shown that (1) -AR stimulation activates NKA in mouse cardiac myocytes by increasing its affinity for internal Na, with no significant effect on the maximum pump rate, (2) PLM inhibits NKA activity by decreasing its [Na]i affinity, and (3) -AR stimulation of NKA is mediated by PLM phosphorylation. Thus, our data indicate that PLM modulates the NKA function in a manner similar to the way PLB affects SERCA.
Acknowledgments
This work was supported by grants from the National Institutes of Health (HL-30077, HL-64724; D.M.B.), the Cardiovascular Research Center at University of Virginia (A.L.T.), and fellowships from the American Heart Association (S.D., J.B.). The authors thank Brian French and Jaime O’Brien for myocyte preparation.
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