Phospholemman Phosphorylation Mediates the Protein Kinase C–Dependent Effects on Na+/K+ Pump Function in Cardiac Myocytes
Because phospholemman (PLM) regulates the Na+/K+ pump (NKA) and is a major cardiac phosphorylation target for both protein kinase A (at Ser68) and protein kinase C (PKC) (at both Ser63 and Ser68), we evaluated whether PLM mediates the PKC-dependent regulation of NKA function and protein kinase A/PKC crosstalk in ventricular myocytes. PKC was activated by PDBu (300 nmol/L), and we measured NKA-mediated [Na+]i decline (fluorescence measurements) and current (Ipump) (voltage clamp). In wild-type mouse myocytes, PDBu increased PLM phosphorylation at Ser63 and Ser68, Ipump (both at 10 and 100 mmol/L Na+ in the pipette solution) and maximal NKA-mediated Na+ extrusion rate (Vmax) from 7.9±1.1 to 12.7±1.9 mmol·L−1 per minute without altering NKA affinity for internal Na+ (K0.5). In PLM knockout mice, PDBu had no effect on either Vmax or K0.5. After pretreatment with isoproterenol (ISO) (1 μmol/L), PDBu still increased the NKA Vmax and PLM phosphorylation at Ser63 and Ser68. Conversely, after pretreatment with PDBu, ISO further increased the Na+ affinity of NKA and phosphorylation at Ser68, as it did alone without PDBu. The final NKA activity was independent of the application sequence. The NKA activity in PLM knockout myocytes, after normalizing the protein level, was similar to that after PDBu and ISO treatment. We conclude that (1) PLM mediates the PKC-dependent activation of NKA function in cardiac myocytes, (2) PDBu and ISO effects are additive in the mouse (affecting mainly Vmax and K0.5, respectively), and (3) PDBu and ISO combine to activate NKA in wild-type to the level found in the PLM knockout mouse.
The autonomic nervous system is an important modulator of heart function. The sympathetic transmitters, epinephrine and norepinephrine, bind to and stimulate adrenergic receptors in the membrane of cardiac myocytes. Stimulation of β-adrenergic receptors (β-ARs) activates protein kinase A (PKA) via adenylyl cyclase–mediated increase in cAMP concentration. The signaling pathway initiated by stimulation of α-ARs involves activation of coupled G proteins (Gq), which in turn cause the activation of phospholipase C (PLC). This leads to the formation of diacylglycerol and activation of protein kinase C (PKC).
The Na+/K+ pump (NKA) is the main pathway for Na+ extrusion from the cells and therefore plays an essential role in the regulation of intracellular Na+ concentration ([Na+]i), which, via the Na+/Ca2+ exchanger, is essential in controlling intracellular Ca2+ and contractility in the heart. There is evidence that sympathetic transmitters can adjust the activity of the NKA to the functional demands of the heart (reviewed previously1,2). We have shown recently3 that β-AR stimulation enhances the NKA activity in mouse ventricular myocytes and the effect is mediated primarily by phosphorylation of phospholemman (PLM), a small transmembrane protein. PLM belongs to a family of proteins (FXYD gene family) that bind to and regulate the NKA in various tissues.4–7 PLM is the only FXYD protein present in cardiac myocytes, where it is a major substrate for both PKA- (at site Ser68) and PKC-dependent (at sites Ser63 and Ser68) phosphorylation.8,9 α-AR stimulation and PKC have been shown to activate the NKA in myocytes from rat and guinea pig,10–13 but the mechanism involved is unclear. Thus, the first aim of this report was to investigate whether PKC-dependent regulation of NKA function is also mediated by PLM. We combined measurements of NKA-mediated [Na+]i decline and NKA current (Ipump) to determine the effect of PKC activation by PDBu (300 nmol/L) on NKA function in ventricular myocytes from mice in which the PLM gene was targeted14 (PLM knockout [PLM-KO]) and wild-type (WT) littermates.
Both PKA and PKC are activated during sympathetic stimulation of the heart. However, a potential crosstalk between PKA- and PKC-dependent regulations of the NKA has not been studied widely. One study found that in guinea pig ventricular myocytes the effects of PKA and PKC stimulation on Ipump were additive and the basal activity of each kinase was involved in the modulation of the pump.10 It is however not known how the signaling pathways for the 2 kinases interact in regulating the NKA. Because PLM modulates the activity of the NKA and can be phosphorylated by both PKA and PKC at Ser68 and by PKC alone at Ser63, it may integrate the PKA- and PKC-dependent effects on the NKA function. Therefore, the second aim of this report was to test whether the sequential effects of PKA and PKC on the NKA are additive, synergistic, or antagonistic and whether phosphorylation of PLM has any role in this possible crosstalk. We determined the effect of successive application of isoproterenol (ISO) (PKA activation) and ISO plus PDBu (PKC activation with PKA still fully activated by ISO) and vice versa on the NKA function and PLM phosphorylation at Ser63 and Ser68 in mouse ventricular myocytes.
Materials and Methods
Generation of PLM-KO mice
PLM-KO mice were generated as previously described,14 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.14 All animal protocols were approved by the animal care and use committees at Loyola University Chicago and University of Virginia.
Mouse ventricular myocytes were isolated as previously described.15 Briefly, hearts were excised quickly after mice were anesthetized (inhalation of 5% isoflurane) and mounted on a gravity-driven Langendorff perfusion apparatus. Hearts were perfused for 8 minutes at 37°C with nominally Ca-free solution with 24 mmol/L Na+HCO3 bubbled with 95% O2:5% CO2 (pH 7.4). Perfusion was then switched to DMEM solution containing 1.27 mg/mL collagenase (type B, Sigma). When the heart became flaccid (≈6 to 8 minutes), ventricular tissue was removed, dispersed, and filtered, and myocytes suspensions were rinsed several times.
Intracellular [Na+] Measurements in Intact Myocytes
Isolated myocytes were plated on laminin-coated coverslips and incubated with 10 μmol/L sodium-binding benzofuran isophthalate/acetoxymethyl ester (SBFI-AM) in the presence of Pluronic F-127 (0.05% wt/vol) for 90 minutes at room temperature. After washing out the external dye, SBFI-AM was allowed to further deesterify for 20 minutes. The normal Tyrode’s solution contained (in mmol/L): 140 Na+Cl, 4 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES, and 10 glucose (pH=7.4). Dual excitation measurements (at 340 and 380 nm; F340 and F380) were performed as previously described.16 F340/F380 was calculated after background subtraction and converted to [Na+]i by calibration at the end of each experiment (using divalent-free solutions with 0, 10, or 20 mmol/L extracellular [Na+]).16 All the measurements in this study were at room temperature.
Na+ Efflux Through the NKA
NKA flux was determined as the rate of pump-mediated [Na+]i decline.16 Myocytes were Na+ loaded by inhibiting the NKA in a K-free solution containing (in mmol/L): 145 Na+Cl, 2 EGTA, 10 HEPES, and 10 glucose (pH=7.4). [Na+]i decline was measured on pump reactivation in solution containing (in mmol/L): 140 tetraethylammonium chloride, 4 KCl, 1 MgCl2, 2 EGTA, 10 HEPES, and 10 glucose (pH=7.4). Because cell volume does not change with this protocol,17 [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=Vmax/(1+(K0.5/[Na+]i)nHill), where Vmax is the maximum Na+ extrusion rate of the pump, K0.5 is the [Na+]i for the half-maximal activation of the pump, and nHill is the Hill coefficient.
Simultaneous NKA Current and [Na+]i Measurements
Simultaneous Ipump and [Na+]i measurements were performed as previously described.18 Briefly, thapsigargin pretreated (1 μmol/L for 10 minutes) myocytes were whole-cell voltage clamped under conditions that minimized the effect of cell dialysis by the patch pipette on [Na+]i (initial pipette resistance of 3 to 5 MΩ) while maximizing its control by transsarcolemmal Na+ fluxes. The standard pipette solution contained (in mmol/L): 10 Na+Cl, 20 KCl, 100 K-aspartate, 20 tetraethylammonium chloride, 10 HEPES, 5 Mg-ATP, 0.7 MgCl2 (≈1 mmol/L free Mg), 3 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetate (BAPTA), 1.15 CaCl2 (≈100 nmol/L free Ca2+), 1 SBFI tetraammonium salt (pH=7.2). The external solution contained (in mmol/L): 136 Na+Cl, 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 −20 mV (to inactivate Na+ channels) as the outward shift induced by switching from K+-free to K+-containing external solution. We showed previously that this current is equivalent to ouabain-sensitive current.18 [Na+]i was measured as above and calibration was done at the end of each experiment.
Ipump Measurements at Saturating [Na+]i
Ipump measurements at saturating [Na+]i (100 mmol/L in the pipette solution, with Na+ replacing K+) were performed using low-resistance electrodes (initial resistance ≤2 MΩ). Myocytes were perfused with K-free external solution after reaching the whole-cell configuration for ≈10 minutes to allow for equilibration of intracellular and pipette Na+. Ipump was then measured at −20 mV as the outward shift induced by switching from K+-free to K+-containing external solution in the absence and presence of PDBu (300 nmol/L). Ipump traces were plotted as a function of recording time.
Isolated myocytes were treated with PDBu (Calbiochem, 300 nmol/L) and then lysed in ice-cold buffer containing 1% Nonidet P-40 and (in mmol/L) 150 Na+Cl, 10 Tris (pH 7.4), 2 EGTA, 50 NaF, 0.2 NaVO3, and protease inhibitors (Calbiochem). Alternatively, myocytes were treated with ISO (1 μmol/L) and/or PDBu for 10 minutes, in some cases, followed by another 10 minutes treatment with ISO or PDBu. Lysates were immediately flash frozen and stored at −80°C. SDS-PAGE and quantitative immunoblotting were performed as previously described.19 Custom-made PLM antibodies (described in Silverman et al20) were used to detect phosphorylation at site Ser63 and Ser68 respectively (CP-63 and CP-68 antibody). Blots were developed using enhanced chemiluminescence (Pierce supersignal west dura substrate). Signals were recorded with an UVP EpiChemi II darkroom imaging system and quantitated with ImageJ software (NIH). Signals were normalized to control sample signals on the same gel and equal protein loading was ensured by reprobing with GAPDH.
Data are expressed as mean±SEM. Statistical discriminations were performed with Student’s t test (paired when appropriate) with P<0.05 considered significant (*).
Effect of PDBu on NKA-Mediated Na+ Efflux in Myocytes From WT and PLM-KO Mice
NKA flux was determined as the rate of pump-mediated [Na+]i decline as previously described.3,16 Myocytes were Na+ loaded by inhibiting the NKA in a K-free solution (Figure 1A). Then the pump was reactivated with K (4 mmol/L, in the absence of external Na+), and [Na+]i decline was measured. The protocol was then repeated in the presence of 300 nmol/L PDBu. In the experiment shown in Figure 1A, Na+ efflux in Na+-free solution is mediated by both NKA and NKA-independent mechanisms. A parallel series of experiments was done to account for the leak 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 NKA-independent component from the total Na+ efflux.
Figure 2A shows average data for the effect of PDBu on the [Na+]i dependence of NKA function in myocytes from WT (7 cells, 4 hearts) and PLM-KO mice (7 cells, 4 hearts). Data were fitted with a Hill expression to derive the Vmax, K0.5, and nHill. In WT mice, PDBu significantly increased Vmax from 7.9±1.1 to 12.7±1.9 mmol·L−1 per minute without any significant change in the pump affinity for internal Na+ (K0.5=18.3±2.7 versus 17.3±3.2 mmol/L). In PLM-KO mice, PDBu had no effect on either Vmax (9.0±0.7 versus 9.0±0.7 mmol·L−1 per minute) or K0.5 (14.3±1.1 versus 12.2±1.1 mmol/L). PDBu did not significantly change nHill in either WT or PLM-KO myocytes (2.8±0.4 versus 2.8±0.3 in WT; 2.8±0.2 versus 3.0±0.2 in KO).
Immunoblots with phosphorylation-specific antibodies (Figure 2B) show a time-dependent increase in PLM phosphorylation at Ser68 and Ser63 in myocytes exposed to PDBu for up to 20 minutes. No PLM was detected in PLM-KO mouse, as indicated by our previous results.3
Effect of PDBu on Ipump in Myocytes From WT and PLM-KO Mice
We determined the effect of PKC stimulation on Ipump by simultaneously monitoring Ipump and [Na+]i under voltage-clamp conditions at a range of relevant [Na+]i.18 For these experiments, we used relatively small patch pipettes (initial resistance was 3 to 5 MΩ, resulting in a series resistance ≥10 MΩ) and larger cells in an attempt to allow the transsarcolemmal Na+ fluxes, rather than the pipette solution to mainly control [Na+]i. This way, we could measure Ipump over a range of [Na+]i (Figure 3). After reaching the whole-cell configuration (allowing the fluorescent indicator to diffuse into the cell), NKA was inhibited for 5 to 10 minutes in an external K-free solution. This resulted in Na+-loading of the myocyte. Then, the pump was abruptly re-activated by restoring external K (4 mmol/L) while measuring membrane current (Ipump, Figure 3A and 3D) and [Na+]i (Figure 3B and 3E). Pump reactivation resulted in a rapid activation of outward Ipump followed by a decay (Figure 3A and 3D), which accompanied the decline in [Na+]i driven by pump function (Figure 3B and 3E). Then, the myocyte was Na+ loaded again in the presence of 300 nmol/L PDBu, followed by measurement of Ipump and [Na+]i on NKA activation with 4 mmol/L external K in the continuous presence of PDBu. Because PDBu enhances Na+ influx (see below), the initial [Na+]i on NKA activation was often different under control and PDBu conditions. However, this does not affect the Ipump versus [Na+]i plot (Figure 3C and 3F) because the plots for 2 consecutive control runs with different initial [Na+]i were superimposable (not shown).
Figure 3A through 3C shows a representative experiment in myocytes from WT mouse. The plot of Ipump versus [Na+]i (Figure 3C) emphasizes that Ipump was significantly higher in the presence of PDBu for a given [Na+]i (0.33±0.04 versus 0.45±0.07 pA/pF at [Na+]i=12.3±0.5 mmol/L, 8 cells, 4 hearts; Figure 3C, inset). In contrast, PDBu had no effect on the [Na+]i dependence of Ipump in myocytes from PLM-KO mouse (Figure 3D through 3F; 0.35±0.05 versus 0.36±0.05 pA/pF at [Na+]i=12.4±0.6 mmol/L, 9 cells, 4 hearts, Figure 3F, inset). Thus, Ipump data are consistent with a PLM-dependent stimulation of NKA function by PDBu. This further confirms that PDBu stimulation of NKA is mediated primarily by PLM.
The PDBu-induced Ipump increase in the experiments from Figure 3 could be attributable to either an increase in Vmax or a decrease in K0.5. To investigate this further, we measured Ipump with 100 mmol/L Na+ in the pipette solution, using low-resistance electrodes (see Materials and Methods), to saturate the pump with respect to internal Na+. In these conditions, PDBu significantly increased Ipump from 1.48±0.06 to 1.62±0.07 pA/pF in myocytes from WT mice and had no effect in the PLM-KO (Figure 4). These results confirm that PDBu increases the Vmax of NKA in a manner that requires PLM.
The extent of Vmax enhancement in WT myocytes in these ruptured patch experiments (Figure 4) was less than in the d[Na+]i/dt experiments (Figure 2). We suspect this is partly because cellular dialysis by the patch pipette solution limits PLM modulation by PKC. This interpretation is consistent with results with higher resistance pipettes as in Figure 3 (where the percentage of Vmax enhancement was higher) and with phospho antibody immunoblots, which showed less PLM phosphorylation when PDBu was applied to saponin-permeabilized myocytes (not shown).
Crosstalk Between PKC- and PKA-Induced NKA Stimulation: Role of PLM
We previously showed3 that β-AR stimulation with ISO (1 μmol/L) activates NKA in WT mouse myocytes mainly by increasing its affinity for internal Na+ and has no effect in PLM-KO mouse. To investigate a possible crosstalk between PKC- and PKA-dependent regulations of the NKA, we determined the effect of PDBu on −d[Na+]i/dt in myocytes that were pretreated with ISO (Figure 5A). PDBu still increased the Vmax of NKA-mediated Na+ extrusion to the same level as in the absence of ISO (Vmax increased from 7.6±0.5 mmol·L−1 per minute with ISO alone to 13.6±0.9 mmol·L−1 per minute with ISO plus PDBu) without a significant change in K0.5 (12.0±0.9 versus 10.6±1.1 mmol/L). Figure 5B shows the converse experiment where after PDBu exposure, ISO further stimulated the NKA function by significantly enhancing the affinity for Na+ (K0.5 decreased from 17.4±1.4 to 11.2±1.2 mmol/L) without further changes in Vmax (11.3±0.6 versus 12.3±0.8 mmol·L−1 per minute). Notably, application of ISO and PDBu resulted in a similar level of NKA stimulation regardless of the application sequence.
The different target of NKA modulation by PKA (K0.5) and PKC (Vmax) activation may be attributable to PLM phosphorylation at different sites: PKA phosphorylates PLM at Ser68, whereas PKC phosphorylates at both Ser68 and Ser63. Figure 5C shows immunoblots with phospho-specific PLM antibodies (for Ser63 and Ser68 phosphorylation sites). ISO increased phosphorylation at Ser68 (2-fold), but not Ser63, whereas PDBu enhanced phosphorylation at both sites (nearly 4-fold at Ser68 and 2-fold at Ser63).
PDBu application after ISO (10 minutes each) increased the Ser63 and Ser68 phosphorylation substantially (Figure 5C). When ISO was applied after PDBu there was further increase in phosphorylation at Ser68. Thus, prior activation of PKA or PKC did not prevent further PLM phosphorylation and stimulation of the NKA by the other kinase. Moreover, dual exposure to PDBu and ISO either simultaneously (for 10 or 20 minutes) or sequentially in either order (for 10 minutes each) raised PLM phosphorylation at both sites to similar apparent maxima (Figure 5C). Functionally, this resulted in a similar level of NKA stimulation.
Effect of PDBu on Resting [Na+]i in Myocytes From WT and PLM-KO Mice
We previously showed that PKA-dependent stimulation of NKA in WT myocytes caused a decline in resting [Na+]i but no effect in PLM-KO myocytes.3 Figure 6 shows the effect of PDBu on [Na+]i in WT and PLM-KO myocytes. In WT, PDBu did not significantly alter resting [Na+]i (Figure 6B; 9 cells from 3 hearts). However, in PLM-KO myocytes PDBu increased [Na+]i by 2.4±0.6 mmol/L (Figure 6B; 13 cells from 3 hearts). Because PKC activation can stimulate the Na+/H exchange,21,22 PDBu may enhance Na+ influx, independent of PLM or NKA. This may explain why PDBu increased resting [Na+]i in PLM-KO myocytes, despite unaltered NKA function (Figure 2A).
We further tested whether PDBu alters Na+ influx by measuring the rate of Na+ rise on NKA inhibition (as in Figure 1A). PDBu enhanced Na+ influx rate in both WT and PLM-KO myocytes (Figure 6C, upper bars) at 12 mmol/L [Na+]i (resting [Na+]i in both WT and PLM-KO mice3). We compared this to the PDBu-induced increase in Na+ efflux, based on NKA function curves in Figure 2A (also at 12 mmol/L [Na+]i; Figure 6C, lower bars). For WT myocytes, the PDBu-induced increase in Na+ influx (2.9±1.1 mmol L−1 min−1) was nearly counter-balanced by the enhancement of NKA activity (2.5±1.2 mmol·L−1 per minute at 12 mmol/L), explaining the unaltered [Na+]i in WT myocyte on PDBu exposure. In PLM-KO mice, the PDBu-induced increase in Na+ influx exceeds the negligible effect on NKA function, resulting in increased [Na+]i (as in Figure 6A). Thus, PKC-dependent NKA activation may compensate for PKC-dependent stimulation of Na+ influx.
The present study shows that PKC stimulates the NKA in mouse ventricular myocytes by increasing its Vmax with no significant effect on the affinity for internal Na+. The absence of NKA stimulation in PLM-KO mouse suggests that PKC-dependent effects on the pump are mediated primarily by PLM, rather than direct NKA phosphorylation. Additionally, PKC and PKA appear to have additive effects on NKA function.
Regulation of the NKA by PKC in Mouse Ventricular Myocytes: Role of PLM
There is some consensus that α-AR or PKC activation enhance the activity of the NKA,10–13 but the mechanism responsible is still unknown. PKC can directly phosphorylate the α subunit of the NKA in intact rat cells, but this does not seem to affect either the Vmax or the apparent Na+ affinity of the pump.23 An alternative mechanism involves phosphorylation of a NKA regulatory protein. We and others have shown previously that PLM, a member of FXYD family of proteins that associate with and modulates the NKA in various tissues, coimmunoprecipitates with the α subunit of the NKA.3–5,7,19,20,24 Moreover, we demonstrated that PLM inhibits the NKA and this inhibition is relieved by PLM phosphorylation on ISO stimulation.3 β-AR stimulation of the NKA was mediated primarily by PLM phosphorylation, as no effect was observed in the PLM-KO mice.3 Studies on shark-like PLM (PLMS) indicated that phosphorylation of PLMS by PKC or selective proteolysis of the C terminus increase the NKA activity.25,26 Here we show that PKC activation with PDBu stimulates the NKA in intact and voltage-clamped mouse ventricular myocytes and the effect is absent in cells from PLM-KO mice. This suggests that PKC-dependent effects on the NKA are also mediated primarily by PLM.
Our data indicate that PKC stimulates the NKA in mouse ventricular myocytes by increasing its maximum Na+ extrusion rate with no effect on the affinity for intracellular Na+. We found a 60% increase in Vmax following PDBu treatment in intact cells (−d[Na+]i/dt experiments). The effect was reduced, but still significant, in ruptured-patch experiments. Vmax increased by 30% when using small pipette (3 to 5 MΩ, series resistance ≥10 MΩ; experiments as in Figure 3) and by only 10% with larger pipettes used to optimize dialysis with high [Na+]i (<2 MΩ, series resistance <5 MΩ; experiments as in Figure 4). This reduction in the PKC effect may be caused by dialysis of PKC in the voltage-clamp experiments. Indeed, immunoblots showed that PLM phosphorylation by both PDBu and ISO was approximately 50% lower at both Ser68 and Ser63 in myocytes permeabilized with saponin compared with intact myocytes (not shown). The rise in Vmax found here is in agreement with data in myocytes from rat and guinea pig that show a higher Ipump at an internal Na+ that likely saturates the pump.10–13 However, prior data on PKC effects on NKA K0.5 are lacking.
We found previously that NKA activation by ISO in mouse is mainly attributable to an increase in the affinity of the pump for internal Na+. However, we could not rule out an effect of ISO on Vmax, which may or may not be mediated by PLM. Both here and in our previous study,3 Vmax under control conditions was 10% to 20% higher in myocytes from PLM-KO versus WT mice, but the difference was not significant. However, the NKA expression level is 20% to 25% lower in PLM-KO myocytes.3 Thus, when normalizing to the protein level, Vmax in PLM-KO mice is ≈50% higher than in WT, which compares well to the rise in Vmax induced by PDBu in intact WT myocytes. Thus, it is likely that PLM inhibits the NKA by reducing both its Vmax and the affinity for intracellular Na+. Whether the relief of inhibition on PLM phosphorylation occurs through Vmax, K0.5 or both may be a function of the phosphorylation site (Ser63 versus Ser68), the NKA α-subunit isoform, or the species.27
Crosstalk Between PKA and PKC Pathways in Activating the NKA
Both PKA and PKC are activated during sympathetic stimulation of the heart. Thus, it is important to determine whether the sequential effects of PKA and PKC on the NKA are additive, redundant, synergistic, or antagonistic. Here we found that the successive application of ISO (1 μmol/L; PKA activation) and PDBu (300 nmol/L; PKC activation with PKA still fully activated by ISO) resulted in a maximum stimulation of the NKA activity in mouse ventricular myocytes. This was paralleled by a near maximum phosphorylation of PLM at both Ser68 (PKA and PKC site) and Ser63 (PKC site). PKA activation (with ISO) after strong PKC activation also further stimulated the NKA and induced appreciable further PLM phosphorylation at Ser68. Thus, the effect of PKA and PKC activation on K0.5 and Vmax, respectively, was independent of the other kinase and could be additive. An interesting possibility is that PKA and PKC regulation target different NKA α-subunits isoforms (or have access to different pools of PLM). Indeed, in guinea pig ventricular myocytes, the β-AR effects on Ipump are targeted to the α1 isoform (and not α2),20,28 whereas α-AR stimulation affects only the α2 isoform.28 The simplest interpretation is appealing; that is, PLM phosphorylation at Ser63 (by PKC) mediates the Vmax enhancement, whereas phosphorylation at Ser68 (by PKA) mediates the enhanced Na+ affinity. However, it must not be as simple as that because the level of Ser68 phosphorylation produced by PKC activation was higher than that produced by ISO, yet the NKA modulation differed. Of course, there could be some complex type of crosstalk between these regulatory phosphorylation sites, but further investigation will be required for clarification.
Physiological Context of PKC Activation of NKA
PKC activation in response to α-AR activation stimulates the Na+/H exchanger to extrude protons in exchange for Na+. As a result, both intracellular pH and [Na+]i will rise,21,22 which leads to an increase in the myofilament Ca2+ sensitivity and Ca2+ transients via Na+/Ca2+ exchange (NCX), respectively. Action potential duration is typically increased by α1-AR activation in parallel to the positive inotropic effect,29 which may be attributable primarily to decreased K currents. The action potential prolongation seen in most myocytes would tend to increase Ca2+ influx (by ICa and possibly NCX) and also decrease Ca2+ efflux via NCX (allowing greater SR Ca2+ uptake). This could increase the likelihood of triggered arrhythmias. Enhancement of NKA activity may thus play a role in the sympathetic response of the heart to enhance Na+ extrusion, which would offset the higher level of Na+ influx (and Ca2+ through NCX). Our data in resting [Na+]i in response to PKC activation fit into this notion that NKA stimulation balances [Na+]i influx in resting myocytes from WT but not PLM-KO mice (Figure 6). PLM may also directly affect NCX function30,31 and PKC activators regulate NCX,32 which may also alter Na+ influx during PDBu application.
In summary, we have shown that (1) PKC-dependent stimulation of the NKA in mouse ventricular myocytes is mediated primarily by PLM phosphorylation and enhancement of Vmax, (2) both PKA and PKC are needed to fully activate the NKA, and (3) PKC- and PKA-induced stimulation of the NKA (by enhancing Vmax and Na+ affinity, respectively) is relatively independent of the other kinase in mouse ventricular myocytes.
We thank Li-Guo Jia for help with genotyping and care of the mice, Brian French and Jayme O’Brien for myocyte preparation, and Kenneth S. Ginsburg for analysis software.
Sources of Funding
This work was supported by NIH grants HL-30077, HL-64724, HL-81562 (to D.M.B.); the Cardiovascular Research Center at the University of Virginia (to A.L.T.); and fellowships from the American Heart Association (to F.H., J.B., S.D.).
Original received April 21, 2006; revision received October 10, 2006; accepted October 26, 2006.
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