Published online before print April 5, 2007, doi: 10.1161/01.RES.0000266742.51389.a4
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© 2007 American Heart Association, Inc.
Cellular Biology |
Regulation of L-Type Calcium Channel and Delayed Rectifier Potassium Channel Activity by p21-Activated Kinase-1 in Guinea Pig Sinoatrial Node Pacemaker Cells
From the Department of Physiology and Biophysics and Center for Cardiovascular Research (Y.K., M.S.B., R.J.S.), University of Illinois at Chicago; Division of Cardiovascular and Endocrine Sciences (M.L.), University of Manchester, UK; and Department of Pharmacology (T.P.C., S.R., P.A.D.M., M.Y., D.A.T.), University of Oxford, UK.
Correspondence to Dr Ming Lei, MD, PhD, Division of Cardiovascular and Endocrine Sciences, University of Manchester, Manchester M13 9XX, UK. E-mail ming.lei{at}manchester.ac.uk
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Abstract |
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Phosphorylation of ion channels plays an important role in the regulation of cardiac function, but signaling mechanisms controlling dephosphorylation are not well understood. We have tested the hypothesis that p21-activated kinase-1 (Pak1), a serine–threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity through a mechanism involving protein phosphatase 2A. We report a novel role of Pak1-mediated signaling in attenuating isoproterenol-induced enhancement of L-type Ca2+ current (ICaL) and delayed rectifier potassium current (IK) in guinea pig SAN pacemaker cells. We demonstrate that in guinea pig SAN: (1) there is abundant expression of endogenous Pak1 in pacemaker cells; (2) expression of constitutively active Pak1 depresses isoproterenol-induced upregulation of ICaL and IK; (3) inhibition of protein phosphatase 2A increases the enhancement of IK and ICaL by isoproterenol in Ad-Pak1–infected cells; (4) protein phosphatase 2A coimmunoprecipitates with endogenous Pak1 in SAN tissue; and (5) expression of constitutively active Pak1 suppresses the chronotropic action of isoproterenol on pacemaker activity of intact SAN preparations. In conclusion, our data demonstrate that a Pak1 signaling pathway exists in cardiac pacemaker cells and that this novel pathway plays a role in the regulation of ion channel activity.
Key Words: p21-activated kinase-1 • pacemaking • sinoatrial node • L-type calcium channels • voltage-dependent potassium channels
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Introduction |
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Signaling pathways activated by ß-adrenoceptors that increase heart rate have been well studied (reviewed elsewhere1–4), but parasympathetic pathways that attenuate this signaling and cause a slowing of heart rate are less well understood. Although there is evidence that the antiadrenergic effect of acetylcholine is achieved at least in part by a depression in adenylate cyclase activity and reduced cAMP levels mediated via inhibitory G proteins,5 emerging evidence suggests an important role for a signaling pathway involving protein phosphatase 2A (PP2A).6,7 This idea fits with evidence indicating a dynamic balance between kinase and phosphatase activity in the control of cardiac ion channel phosphorylation and dephosphorylation even in the absence of autonomic stimulation.8–11 Pathways regulating the phosphorylation of ion channels in cardiac cells are relatively well understood,12 but possible signaling pathways controlling dephosphorylation remain unclear.
We have tested the hypothesis that signaling through p21-activated kinase-1 (Pak1), a serine–threonine protein kinase regulated by Ras-related small G proteins, regulates sinoatrial node (SAN) ion channel activity. Our hypothesis is based on our demonstration that Pak1 binds to PP2A and induces dephosphorylation of cardiac myofilament proteins.7 Results of our approach, which involved infection of SAN pacemaker cells with an adenovirus expressing constitutively active Pak1 (Ad-Pak1), support this hypothesis.
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Materials and Methods |
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Animals
Age-matched (12-week-old) female guinea pigs were purchased from Charles River (Wilmington, Mass; Margate Kent, UK); procedures were performed in accordance with national and institutional guidelines (US and UK).
Viral Infection
Freshly isolated guinea pig SAN pacemaker cells were prepared by procedures based on previous methods13 and were allowed to settle onto laminin-coated coverslips in 6-well tissue culture plates. These cells were cultured for 2 hours in RPMI medium 1640+4% FBS (Invitrogen, Paisley, UK) for cell attachment. Fresh medium (RPMI medium 1640+4% FBS) containing Ad-Lacz or Ad-Pak1 was added to the plates at a multiplicity of infection of 100. Ad-Pak1 (expressing constitutively active Pak1) and Ad-Lacz were generated as described.7 In the case of infection of intact SAN tissue, Ad-Pak1 was introduced into the nodal region by using Langendorff perfusion of the heart. Histamine (10 mmol/L) was included in the perfusion solution to increase the efficiency of viral delivery. Dissection of the intact SAN preparations was performed as previously described.13 The SAN was dissected after 30 minutes of perfusion with Tyrode solution containing Ad-Pak1 or Ad-Lacz viruses (109 pfu) and superfused with Tyrode solution for 6 hours for later characterization of electrophysiological function. Expression of the recombinant Pak1 in SAN was detected by immunofluorescence with an antibody (Ab) that specifically recognized an hemagglutinin (HA) tag on the expressed Pak1. Recombinant adenovirus that expresses ß-galactosidase (Ad-Lacz) was used as a control. The activity of the SAN was monitored over a 6-hour period by extracellular potential recording, as we described previously.13
Western blotting and immunoprecipitation for determining the expression of endogenous Pak proteins and interaction of Pak1 with PP2A in freshly isolated and cultured pacemaker cells were performed as we described previously.7
Immunocytochemistry and Imaging With Confocal Microscopy
Single immunocytochemical staining was performed as we described previously.13 In the case of costaining, to determine the interaction of active-Pak1 and PP2A in Ad-Pak1–transfected cells, methods for cell culture and gene transfer were as described above in the section on viral infection. Cells were fixed with 4% paraformaldehyde/PBS for 10 minutes, after which they were washed 3 times with PBS for 10 minutes and permeabilized with 0.1% Triton X-100/PBS for 20 minutes. Cells were then washed 3 times with PBS for 10 minutes and incubated with 1% donkey serum albumin/PBS blocking buffer for 60 minutes. Following removal of blocking buffer, cells were incubated overnight with 1% donkey serum albumin/PBS containing both rabbit anti-HA (1:100) and mouse anti-PP2A Abs (1:100). Cells were then washed 3 times with PBS for 10 minutes and incubated in the dark with the secondary Abs (1:400): goat anti-rabbit IgG conjugated to Alexa Fluor 488 (Molecular Probes, Eugene, Ore) and donkey anti-mouse IgG conjugated to Cy3 (Chemicon). Cells were then washed 3 times with PBS for 10 minutes in the dark, and the coverslips were mounted on slides with fluorescent mounting medium (DakoCytomation, Glostrup, Denmark). The slides were viewed on a scanning laser inverted microscope (LSMZ1, Carl Zeiss Inc) equipped with argon and helium–neon lasers, which allowed excitation at 488 and 568 nm wavelengths for the detection of fluorescein isothiocyanate/Alexa 488 and Cy3, respectively. To achieve an optimal signal-to-noise ratio for each fluorescence signal, we scanned sequentially with 568 and 488 nm.
Electrophysiology Recording
Patch-clamping was performed using the amphotericin-perforated patch technique.13 Tyrode solution contained (in mmol/L): NaCl, 130; KCl, 5.4; CaCl2, 1.8; MgCl2, 3.5; NaH2PO4, 0.4; glucose, 10; taurine, 20; HEPES, 5; titrated to pH 7.4 with NaOH. The pipette solution used for recording of L-type Ca2+ channel current (ICaL) contained (in mmol/L): KCl, 140; MgSO4, 1.8; HEPES, 5; Na+-EGTA, 5; titrated to pH 7.3 with KOH. For recording of delayed rectifier K+ current (IK), the pipette solution contained (in mmol/L): KCl, 150; NaCl, 5; MgCl2, 2; K2ATP, 1; HEPES, 5; titrated to pH 7.3 with KOH. In the case of extracellular potential recording from isolated SAN preparations, the preparation was superfused with Tyrode solution at 35°C at a rate of 4 mL min–1 via a heat exchanger. Extracellular potentials were recorded by bipolar electrodes as we described previously.13 Electrical signals were digitized at 5 kHz by a DigiData 1322A A/D converter (Axon Instruments Inc) and stored on a computer for later analysis.
Antibodies
Antibody preparations were as follows: Pak1 Ab (Santa Cruz Biotechnology), 1:50 (dilution) for immunofluorescence and 1:500 for Western blotting; anti-HA Ab (Sigma), 1:100 for immunofluorescence and 1:1000 for Western blotting; PP2A Ab (Upstate), 1:100 for immunofluorescence 1:1000 for Western blotting.
Data Analysis
All data passed normality and equal variance tests. Analysis was performed using the paired or unpaired Student’s t test as appropriate (SigmaStat, Systat Software Inc), and significance was accepted at P<0.05. Data are presented as mean±SE.
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Results |
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Endogenous Pak Proteins and Constitutively Active Pak1 (HAPak1) in the SAN
We first characterized the expression of Pak in the SAN, which has not been reported previously. Our observations using Western blot techniques show that the Pak1 protein is abundant in SAN, atrial, and ventricular tissue (Figure 1B). Pak2 is expressed at low levels, and there is little expression of Pak3 (Figure 1A). In immunohistochemical experiments using confocal microscopy, we found endogenous Pak1 to be localized in SAN cells in distinct subcellular distribution patterns. Figure 1C and 1D shows a typical pattern of expression of endogenous Pak1 (
100 cells were examined). Pak1 was evenly expressed in small cells (presumably from the SAN center) (Figure 1C), whereas Pak1 labeling demonstrated clear striations in large cells (presumably from the periphery) (Figure 1E), with a mixed pattern in medium size cells (Figure 1D).
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To investigate the functional role of Pak1 in the SAN, we infected SAN pacemaker cells with constitutively active Pak1 (Figure 2A). The transfection efficacy was determined with fluorescence microscopy using the adenovirus Ad-eGFP; Ad-Pak1 expression was tested by immunolabeling with an Ab against the HA tag. Expression began at 4 to 6 hours and peaked at 12 hours. Figure 2B shows the expression of control virus Ad-eGFP at 12 hours. In contrast to endogenous Pak1, HAPak1 tended to concentrate in a patched pattern in pacemaker cells (Figure 2C). In further experiments to assess the functional effect of Ad-Pak1 on SAN pacemaker activity, infection of Ad-Pak1 in intact SAN preparations was obtained by Langendorff perfusion with adenoviral vectors (ie, Ad-eGFP, Ad-Lacz, and Ad-Pak1). Expression of HAPak1 was detected in SAN tissue sections by immunostaining of anti-HA, although expression was not detected in all cells (with approximately 20% to 30% showing expression; see Figure 7A).
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Effect of Constitutively Active Pak1 on ICaL and IK in Pacemaker Cells
An important question is whether Pak1 is involved in signaling pathways that regulate ion channel activity by dephosphorylation in cardiac myocytes, and, in particular, whether this is the case in the pacemaker cells that are the focus of this study. As proof-of-concept, we investigated the effects of constitutively active Pak1 on ICaL and IK; these 2 ion channel pathways are major players in generating SAN pacemaker activity and are targets of cAMP/protein kinase A (PKA) signaling in the regulation of SAN pacemaker rhythm. The pacemaker cells were identified by their specific morphology (spindle or spider shape) and, in the case of patch clamping experiments, by cell capacitance (because the total surface membrane capacitance of SAN pacemaker cells is between 15 and 50 pF).
ICaL was activated by 200-ms step depolarizations to potentials between –40 and +50 mV from a holding potential of –50 mV. NiCl2 (40 µmol/L), E-4031 (1 µmol/L), and tetrodotoxin (20 µmol/L) were added to the external solution to block, respectively, T-type Ca2+ current (ICaT), the rapid component of the IK, and the fast Na+ current. Basal ICaL in cells from both groups was statistically indistinguishable (Ad-Lacz, –15.0±3.0 pApF–1 [Cm, 32±2 pF; range, 28 to 40 pF; n=7]; versus Ad-Pak1, –13.0±1.0 pApF–1 [Cm, 34±5 pF; range, 28 to 42 pF; n=6; P>0.05]) (Cm is cell capacitance; Figure 3A through 3D). In the case of the control Ad-Lacz group, ICaL was significantly enhanced in the presence of 100 nmol/L isoproterenol (ISO): the peak current at 0 mV increased from –15.0±3.0 to –28.0±1.3 pApF–1 (P<0.01; n=7) (Figure 3A, 3C, and 3E), and current amplitude increased by 57±6%. However, the effect of 100 nmol/L ISO on ICaL in the Ad-Pak1 group was much less (an increase in peak current at 0 mV by 13±3% from –13.0±1.0 pApF–1 in the absence of ISO to –16.0±1.4 pApF–1 in the presence ISO; P<0.05; n=6) (Figure 3B, 3D, and 3E). This difference in response to ISO indicates that the ISO-induced increase in ICaL in cells infected with Ad-Pak1 was significantly attenuated.
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IK was activated by either 1-second step depolarizations to a range of potentials between –40 and +50 mV, or by a single step depolarization to +40 mV, from a holding potential of –40 mV. IK was measured as the magnitude of the tail current following repolarization to the holding potential. IK measured following repolarization from a step to +40 mV was statistically indistinguishable in cells from both groups: Ad-Lacz, 1.2±0.3 pApF–1 (Cm, 37±4 pF; range, 20 to 49 pF; n=6); versus Ad-Pak1, 1.9±0.5 pApF–1 (Cm, 35±3 pF; range, 18 to 44 pF; n=7) (Figure 4A through 4
F). In the control Ad-Lacz group, IK amplitude was significantly enhanced by 100 nmol/L ISO: the tail current following repolarization from a step to +40 mV was increased by 409±62%, from 1.2±0.3 to 5.0±1.0 pApF–1 (P<0.01; n=6) (Figure 4E and 4F). However, the effect of ISO on IK in the Ad-Pak1 group was again much less than that of the control group: 100 nmol/L ISO induced an increase in IK (following a step to +40 mV) of 69±21%, from 1.9±0.5 pApF–1 in the absence of ISO to 2.6±0.5 pApF–1 in the presence of ISO (P<0.05; n=7) (Figure 4E through 4G). It therefore appears that, as was the case with ICaL, the ISO-induced increase in IK was markedly attenuated in cells infected with Ad-Pak1.
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The effect of ISO on the deactivation of IK was also examined in Ad-Lacz and Ad-Pak1 cells. Figure 4G through 4
I shows examples of the decay of IK in Ad-Pak1 and Ad-Lacz cells in the absence and presence of 100 nmol/L ISO; in these experiments, single step depolarization to +40 mV was made from a holding potential of –40 mV. The time constant of IK decay was measured by fitting the tail current (on repolarization to –40 mV) with a single exponential fitting equation (Clampfit 9.2, Axon). In the control Ad-Lacz group, the rate of IK decay was significantly increased by 100 nmol/L ISO: the time constant of the decay (
) was reduced by 39±8% from 214±9.2 ms to 130±15 ms (P<0.05; n=5). In contrast, there was no significant effect of ISO on the rate of IK decay in the Ad-Pak1 group: the apparent decrease in the time constant of IK decay in the presence of ISO was 17±9%, from 259±44 ms to 204±36 ms (P>0.05; n=6). It therefore appears that the effects of ISO on IK decay were markedly attenuated in Ad-Pak1 cells.
Association of Pak1 and PP2A
Another key question concerns the mechanism by which active Pak1 regulates L-type Ca2+ channel (as well as delayed rectifier K+ channel) activity. Based on our previous study on rat ventricular myocytes,7 we hypothesized that the effects of Pak1 overexpression are likely to be related to increased phosphatase activity, reducing the ß-adrenoceptor–mediated stimulation of ion channel activity in SAN pacemaker cells (ICaL and IK in the case of this study). Figure 5A demonstrates that the catalytic subunit of PP2A coimmunoprecipitated with Pak1. As shown in Figure 5B through 5J, immunocytochemistry using confocal microscopy demonstrated association of HAPak1 (constitutively active) and PP2A in cultured SAN and atrial cells.
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The hypothesis was further tested by studying the effect of okadaic acid (OA), a phosphatase inhibitor, on the effects of ISO on ICaL and IK in Ad-Pak1–infected cells. Figure 6 shows an example of this type of experiment. The effects of 100 nmol/L ISO on ICaL and IK in Ad-Pak1–infected cells was first examined in the absence of OA; following wash (to reverse the effects of ISO) for 10 minutes, the cells were superfused with 100 nmol/L OA for 10 minutes to inhibit PP2A activity, and then 100 nmol/L ISO was reapplied to the cells to examine the response of ICaL and IK under conditions of PP2A inhibition. As shown in Figure 6A, 6B, 6F, and 6G, the effect of ISO (in the absence of OA) on ICaL and IK in Ad-Pak1–infected cells was small: the amplitudes of ICaL and IK were increased by only 11% (step to 0 mV) and 75% (following repolarization from +40 mV), respectively. Following inhibition of PP2A by 100 nmol/L OA, the responsiveness to ISO in the Ad-Pak1–infected cells was substantially increased (Figure 6D and 6I): the amplitudes of ICaL and IK were increased by 35% (step to 0 mV) and 213% (following repolarization from +40 mV). Similar observations were made in 3 other cells for IK and 2 further cells for ICaL.
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Positive Chronotropic Effects of ISO in Ad-Pak1–Transfected Intact SAN Preparations
The observations presented above suggest a role for Pak1 signaling in the regulation of cardiac pacemaker activity. Consistent with this are the results from an experiment illustrated in Figure 7, which demonstrates the effect of active Pak1 on the chronotropic response of the SAN to ISO following gene transfer of either Ad-LacZ (as a control) or Ad-Pak1 to the intact SAN using Langendorff perfusion. Figure 7A shows examples of nodal region images obtained from intact node preparations transfected with Ad-Pak1, using immunohistochemistry techniques with an Ab against the HA tag. Distinct regions of transfected cells were observed across the whole nodal region with a nonuniform pattern; only approximately 20% to 30% of cells were labeled. Figure 7B shows the chronotropic response of SAN preparations infected with Ad-LacZ or Ad-Pak1 (6 hours after gene transfer) to ISO over a range of concentrations (1 nmol/L to 1 µmol/L); extracellular potentials were recorded from the center of the SAN. In these experiments, application of ISO (1 nmol/L to 1 µmol/L) caused a concentration-dependent increase in beating rate in both Ad-LacZ and Ad-Pak1–transfected SAN preparations (Figure 7B and 7C), but the chronotropic response in Ad-Pak1–transfected preparations (n=7) (Figure 7B and 7C) was significantly less than that of Ad-LacZ–transfected preparations (n=7) (Figure 7B and 7C) for most ISO concentrations (Figure 7C). For example, the firing rate was increased by 74±4% from 228±5 min–1 under basal conditions to 378±4 min–1 at a concentration of 1 µmol/L ISO in the Ad-LacZ group (n=7), whereas, in the Ad-Pak1 expressing group, the same concentration of ISO caused an increase in firing rate of 52±3% from 224±3 min–1 to 337±3 min–1 (n=7). Thus, expression of Ad-Pak1 attenuated the positive chronotropic actions of ISO.
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Discussion |
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The data presented here are the first to demonstrate expression and functional effects of Pak1 in SAN pacemaker cells. Our data are also novel in demonstrating alterations of L-type Ca2+ and delayed rectifier K+ channel activities when cellular Pak1 activity was enhanced by expressing constitutively active Pak1 in SAN pacemaker cells. The regulatory role of active Pak1 on ICaL and IK is likely attributable to increased activity of the phosphatase PP2A.
Expression of Pak Proteins in the SAN
Although expression of Pak proteins has been reported in various tissues, including the heart, since they were first identified in brain tissue 12 years ago by Manser et al,14 they have not been reported to date in cardiac pacemaker tissue. Our data indicate that Pak1 is the major Pak isoform expressed in SAN cells, although a low level of Pak2 may also be present in this tissue (Figure 1). The expression level of Pak1 in the SAN is similar to that in atria and ventricles (Figure 1). This is consistent with our recent study in rat ventricular myocytes.7 Further characterization of the localization of endogenous Pak1 in SAN cells by immunolabeling revealed distinct subcellular distribution patterns in these cells. A similar striated pattern has been shown in our recent study in rat ventricular myocytes, and this is thought to be attributable to localization of Pak1 to the Z-disc. It is known that in the center of the SAN, the cells (typical nodal cells) are smaller and also relatively "empty" in morphological appearance. It is thought that this is because the myofilaments, mitochondria, and sarcoplasmic reticulum may be more sparse and less well organized in these cells, whereas the cells from the periphery are larger with more abundant myofilaments, giving rise to more pronounced striations (reviewed elsewhere4). The organizational differences between these 2 types of SAN cell may explain the difference in endogenous Pak1 expression in these cells. It remains unclear why active Pak1 has a distinct intracellular localization as compared with the endogenous protein in SAN cells. A prominent change in the Pak1 protein is associated with autophosphorylation. When serine 21 of Pak1 is autophosphorylated, the region surrounding this amino acid loses the ability to bind to Nck.15 In SAN cells, expression of active Pak1 (HAPak1) is not evenly distributed but demonstrates a "patchy" localization. This suggests that when Pak1 is activated, it may associate with a different set of binding partners. Identification of its new partners after kinase activation in SAN cells will help in further defining Pak1 function in the regulation of pacemaker activity.
Effect of Active Pak1 on ICaL and IK
An important question is whether Pak1 is involved in signaling pathways in which dephosphorylation is thought to reduce activation of ion channels in pacemaker cells. ISO (100 nmol/L) increased the amplitudes of ICaL by 57% and IK by 410% in Ad-LacZ–infected cells, whereas the corresponding increases were only 13% and 70% in the Ad-Pak1–infected cells. Moreover, the IK decay time constant was reduced (by 39±8%; Figure 4G through 4I). However, the effect of ISO on the rate of IK decay in the Ad-Pak1 group was much less than that of the control group, and indeed was not statistically significant (17±9%). Such a difference in the response to ISO between the 2 groups indicates that the effects of ISO on ICaL and IK was significantly attenuated in cells infected with Ad-Pak1. This effect of active Pak1 mainly presented when ICaL and IK activity was enhanced by ß stimulation. It is still an open question as to whether active Pak1 affects basal ICaL and IK activity in SAN cells; in our experiments, we did not observe a significant difference between Ad-Pak1– and Ad-Lacz–transfected cells.
The effect of active Pak1 on ICaL and IK in pacemaker cells indicates a role for Pak1 signaling in regulation of cardiac pacemaker activity. Our observations on isolated SAN preparations (Figure 7) strongly support this hypothesis. Our data demonstrate an effect of active Pak1 on the chronotropic response of intact SAN preparations to ISO, following gene transfer of Ad-LacZ or Ad-Pak1 to the cells using Langendorff perfusion. The positive chronotropic response of Ad-Pak1–transfected SAN preparations to ISO was significantly less than that of Ad-Lacz–transfected preparations at most concentrations of ISO tested (Figure 7). Thus, constitutively active Pak1 attenuated the chronotropic response to ISO in Ad-Pak1 group. The extent of the suppression of ISO-induced chronotropy by active Pak1 in these intact SAN preparations was not as great as we had expected, based on our observed effects on 2 major pacemaker currents, ICaL and IK, in isolated SAN cells. This is likely attributable to: (1) a lower transfection efficacy in intact node preparations (only 20% to 30% of cells transfected; Figure 7A) than in single cells (100%); viral infection takes only a few minutes, whereas expression of the transgene takes substantially longer, hence, the different infection efficiency between the 2 models is attributable to the different routes of infection; and (2) the nature of complex mechanisms involving regulation of SAN pacemaking in the intact node.
One possibility that must be considered is that changes in firing rate in the intact SAN preparation may be caused by effects of transfected Pak1 on autonomic nerve varicosities. However, we feel that this is unlikely to be a major contributing mechanism for a number of reasons. In previous experiments that we have performed on SAN preparations, we have found that ß-adrenoceptor and muscarinic receptor antagonists have little or no effect on firing rate (data not shown), consistent with there being only a low level of spontaneous release of noradrenaline or acetylcholine under these conditions. In addition, our observations of effects of Pak1 transfection on ion channel activity in isolated SAN cells, ie, in the absence of any potential influence of autonomic nerves, support the suggestion that the effects of Pak1 are attributable to direct actions of Pak1 in the pacemaker cells themselves. Hence, it is highly likely that the observed actions of Pak1 transfection in whole SAN preparations are the consequence of direct effects on SAN pacemaker cells, rather than on transmitter release from autonomic varicosities.
Association of Pak1 and PP2A
Accumulating evidence indicates that active modulation of Ca2+ influx through L-type Ca2+ channels results from a coordinated interplay between the activities of kinases and phosphatases, even in the absence of humoral stimulation. For example, in the well-known ß-adrenergic receptor/PKA cascade, inhibitor-1 is a downstream PKA target and is activated to attenuate PP1 activity.16 Santana et al17 provided evidence that the phosphatase calcineurin opposes the action of PKA in mouse ventricular myocytes. Application of serine/threonine phosphatase inhibitors alone is sufficient to increase whole-cell ICa in both guinea pig11,18 and mouse8 ventricular myocytes. Application of the PP2A inhibitor OA can activate L-type Ca2+ channels.9 Calyculin A, which inhibits both phosphatases PP1 and PP2A, increases contractility in ventricular myocytes by increasing L-type Ca2+ channel activity.8 In a complementary study, duBell et al reported that addition of exogenous PP2A decreased ICa in rat ventricular myocytes.19 Our immunoprecipitation studies indicated that Pak1 and PP2A form a complex, which suggests that Pak1 may regulate the activity of L-type Ca2+ and K+ channels through the phosphatase PP2A in SAN pacemaker cells. This hypothesis was further tested by studying the influence of OA on the effects of ISO on ICaL and IK in Ad-Pak1–infected cells. As presented above, OA partially reversed the suppressing effect of active Pak1 on the response of ICaL and IK to ISO in Ad-Pak1–infected cells. This effect is presumably through the inhibition of PP2A activity. Therefore, we suggest that the regulatory role of Pak1 is likely attributable to increased phosphatase activity, although, at this stage, we cannot completely exclude other possible pathways. It therefore appears that there is a dynamic balance between kinase and phosphatase activity to control L-type Ca2+ channel as well as delayed rectifier K+ channel activity in SAN pacemaker cells. The balance between these kinase and phosphatase actions may be of importance in controlling cardiac pacemaker activity in response to autonomic and humoral stimulation. The importance of this balance is highlighted by the recent report by Vinogradova et al20 of a high basal PKA-dependent phosphorylation in SAN pacemaker cells that drives rhythmic internal Ca2+ store oscillations and spontaneous beating of these cells. Exploring such dynamic regulatory processes and mechanisms of balance between kinase and phosphatase activity in cardiac pacemaker cells will no doubt provide an insight into the crucial mechanisms that regulate cardiac pacemaking activity under physiological and pathological conditions and should be undertaken in the future.
In summary, data presented here are the first to demonstrate expression and functional effects of Pak1 in SAN pacemaker cells. Alteration of L-type Ca2+ channel and delayed rectifier K+ channel currents occurred when Pak1 activity was enhanced by expressing constitutively active Pak1 in SAN pacemaker cells. It seems likely that the regulatory role of Pak1 on L-type Ca2+ and delayed rectifier K+ channels is attributable to increased activity of phosphatase PP2A.
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Acknowledgments |
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We thank Prof Mark Boyett for his support.
Sources of Funding
This work was supported by NIH grants HL-22231 and HL-64035 (to R.J.S.), The Wellcome Trust (to M.L. and D.A.T.), and the British Heart Foundation (to D.A.T.).
Disclosures
None.
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Footnotes |
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*Both authors contributed equally to this work.
Original received December 5, 2005; resubmission received September 22, 2006; revised resubmission received March 27, 2007; accepted March 28, 2007.
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