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(Circulation Research. 2003;92:897.)
© 2003 American Heart Association, Inc.

Cellular Biology

Protein Kinase A Hyperphosphorylation Increases Basal Current but Decreases ß-Adrenergic Responsiveness of the Sarcolemmal Na⁺-Ca²⁺ Exchanger in Failing Pig Myocytes

Shao-kui Wei, Abdul Ruknudin, Stephen U. Hanlon, John M. McCurley, Dan H. Schulze, Mark C.P. Haigney

From the Division of Cardiology (S.W., S.U.H., J.M.M., M.C.P.H.), Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Md; Department of Microbiology and Immunology (A.R., D.H.S.), University of Maryland, School of Medicine, Baltimore, Md.

Correspondence to Mark C.P. Haigney, MD, Division of Cardiology, Department of Medicine, Uniformed Services University of the Health Sciences, A3060, USUHS, 4301 Jones Bridge Rd, Bethesda, MD 20814. E-mail MCPH{at}aol.com

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
The sodium-calcium exchanger (NCX) protein is the major cardiac calcium extrusion mechanism and is upregulated in heart failure (HF). NCX expression level and functional activity as regulated by ß-adrenergic receptor (ß-AR) stimulation in swine with and without tachycardia-induced heart failure were studied. The Ni²⁺-sensitive NCX current was measured in myocytes from HF and control animals in the basal state or in the presence of isoproterenol, forskolin, 8-Br-cAMP, okadaic acid, or protein phosphatase type 1. Western blot analysis revealed a significant increase in both the 120-kDa (29%) and 80-kDa (69%) fragments in HF (P<0.05 versus control). Despite this modest increase in protein, the basal peak outward NCX current was increased almost 5-fold in HF (P<0.05 versus control). Stimulation with isoproterenol, however, increased the control currents to a significantly greater extent than HF (500% increase in control versus 100% increase in HF, P<0.01); peak stimulated current was not different in HF and control. This reduction in responsiveness to ß-AR stimulation was refractory to forskolin, 8-Br-cAMP, or okadaic acid stimulation. In vitro protein kinase A back-phosphorylation revealed higher phosphorylation capacity of NCX protein in control versus HF, consistent with increased phosphorylation in vivo (hyperphosphorylation) in HF. Protein phosphatase type 1 exposure resulted in a significant reduction (73%) in peak basal current in HF (compared with no significant difference in controls), confirming that the increased basal NCX current in HF is predominately attributable to hyperphosphorylation. NCX expression and activity are thus increased in HF, although ß-AR responsiveness is decreased because of NCX hyperphosphorylation.

Key Words: Na⁺-Ca²⁺ exchange • ß-adrenergic receptor • protein kinase A phosphorylation • protein phosphatase

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The Na⁺-Ca²⁺ exchanger (NCX) is one of the essential regulators of Ca²⁺ homeostasis in cardiac myocytes and plays an important role in Ca²⁺ handling during excitation-contraction (E-C) coupling. The exchanger is capable of transporting 3 Na⁺ for 1 Ca²⁺ in either direction across the sarcolemma, depending on membrane potential and the transmembrane gradients of Na⁺ and Ca²⁺. Although the NCX is the primary mechanism for extruding Ca²⁺ that enters through the L-type Ca²⁺ channel during systole, it may also trigger Ca²⁺ transients under certain conditions.^1–3 Recently, several studies have reported increased NCX expression or activity in animal models of hypertrophy and heart failure (HF) as well as in human HF.^4–6 Despite intense scrutiny, it remains unclear whether the increase in NCX represents an important compensatory mechanism for decreased sarcoplasmic reticular Ca²⁺-ATPase (SERCA) activity or whether it results in a detrimental shift in phenotype. Increased NCX activity could contribute to contractile dysfunction and destabilize action potential repolarization with early or delayed afterdepolarizations (EADs/DADs), thereby triggering fatal ventricular arrhythmias.^7,8

ß-adrenergic receptor (ß-AR) activation is one of most important regulators of beat-to-beat cardiac function. ß-AR stimulation acts via cAMP-dependent protein kinase A (PKA) to phosphorylate functional proteins such as Ca²⁺ channels, SERCA, ryanodine receptor (RyR), and myofibrils^9–11; recently, studies have reported that NCX activity is also regulated by ß-AR stimulation.¹² Despite the fact that norepinephrine levels are consistently augmented in HF,¹³ blunted ß-AR responsiveness manifested by a reduction in contractile reserve has been reported. This phenomenon has been attributed to the downregulation and desensitization of ß-AR receptors in HF.¹⁴ An additional mechanism of reduced ß-AR receptor responsiveness has been described; recent studies have reported that the L-type Ca²⁺ channel and RyR are tonically phosphorylated (hyperphosphorylated) at baseline in failing human myocytes, ^15–18 which could cause altered Ca²⁺ handing and decreased ß-AR responsiveness. However, it is unknown whether the ß-AR regulation of the NCX is altered in HF or what role PKA phosphorylation may have in modulating NCX activity in HF.

Using a pacing-induced failing pig model, we demonstrate in the present study that HF increased NCX expression and activity yet decreased responsiveness of NCX to ß-AR stimulation. This effect is not attributable to reduced ß-AR receptor number or downregulated adenylyl cyclase activity but is related to a tonically enhanced exchanger phosphorylation state, which may be attributable to decreased phosphatase activity.

	Materials and Methods

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Pacing-Induced Pig Heart Failure Model and Cardiomyocyte Isolation and Culture
Induction of heart failure and ventricular cardiomyocyte isolation and culture were carried out as described previously,²⁷ using protocols approved by the university’s Animal Care and Use Committee. In brief, Yorkshire pigs (Archer Farms, Darlington, Md) of either sex between 7 and 10 weeks of age were anesthetized with thiopental sodium (10 mg/kg, IV) to allow tracheal intubation and then maintained with isoflurane. The right external jugular vein was isolated and cannulated. A 58-cm active-fixation pacing lead was advanced under fluoroscopy to the apex of the right ventricle. Adequate positioning was confirmed by pacing threshold and R-wave sensing, and the lead was sutured to underlying fascia. After 24 to 48 hours of recovery, pacing was initiated at 200 beats per minute. All animals underwent transthoracic echocardiography every 5 days until they developed severe systolic dysfunction, as defined prospectively by a fractional shortening <0.16 (>2 SD below the mean fractional shortening seen in the controls, 0.30±0.07, observed in 27 normal animals). On the development of significant left ventricular dysfunction, animals were euthanized with pentobarbital sodium. The hearts were harvested by left lateral thoracotomy and immersed in ice-cold saline. The region of ventricle perfused by the left anterior descending coronary artery was excised, cannulated, and perfused at 15 mL/min for 10 minutes with nominally Ca²⁺-free modified Tyrode’s solution (in mmol/L, NaCl 138, KCl 4, MgCl₂ 1, NaH₂PO₄ 0.33, glucose 10, and HEPES 10 [pH 7.3 with NaOH] at 37°C and oxygenated with 100% O₂). Perfusion was continued for 12 minutes with the same solution but with added 0.24% (wt/vol) collagenase type I (Sigma) and 0.028% protease XIV (Sigma) and then for 10 minutes with washout solution with 0.1 mmol/L CaCl₂ and 0.02% albumin. Sections of well-digested ventricular tissue from the midmyocardial layer of the ventricle were dissected out, and cells were mechanically dissociated and resuspended in buffers of gradually increasing [Ca²⁺]. To remove dead myocytes and residual contaminating cell types, the myocyte suspension was centrifuged through a discontinuous Percoll gradient, usually resulting in >90% rod-shape cells. To allow the myocytes to recover from enzymatic digestion, the cells were cultured overnight at 37°C in serum-free medium 199 supplemented with 5 mmol/L carnitine, 5 mmol/L creatine, 5 mmol/L taurine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 0.25 µg/mL amphotericin. We have found that this approach increases the rate of successful giga-seal formation without significantly altering their phenotype with regard to NCX function.

Western Blot and In Vitro Phosphorylation
A portion of the left ventricle from the same hearts used for electrophysiologic study was frozen in liquid nitrogen at the time of euthanasia and stored at -80°C. Frozen tissue samples were pulverized with a mortar and pestle and then homogenized in immunoprecipitation buffer (5 mL/g) containing (in mmol/L) Triton X-100 50, NaCl 190, MgCl₂ 0.1, HEPES 50, and EDTA 6, pH 7.4, with added proteolytic inhibitors PMSF 1 mmol/L, 1,10-phenonthroline 2 mmol/L, leupeptin 4 µmol/L, pepstatin 1 µmol/L, aprotinin 1000 U/mL, and iodoacetamide 0.2 mmol/L. Undissolved cell fragments were removed by centrifugation at 100g for 10 minutes at 4°C. Supernatant was concentrated 3-fold using Microcon-50 (Millipore), and the total protein concentration was assayed (Bio-Rad Protein Assay kit). The same amount of concentrated samples were mixed with NCX antibody and immunoprecipitated at 4°C overnight. Protein A Sepharose beads were then added and incubated in 4°C for 4 hours. The antigen-antibody complex was washed 3 times with a buffer containing (in mmol/L) Triton-X100 50 (0.1%), NaCl 150, MgCI₂ 0.1, HEPES 50, and EDTA 6, pH 7.4. Half of the sample was used for the Western blotting, and the rest for in vitro phosphorylation. In vitro PKA phosphorylation was performed by incubation of sample with 1 µg of the catalytic subunit of PKA (PKA-CS reconstituted in 5 mmol/L dithiothreitol) and 10 µCi of [-³²P] ATP (3000 Ci mmol/L) in phosphorylation buffer for 10 minutes at 37°C. The reaction was stopped by adding 1 mL of stop buffer (in mmol/L, sodium phosphate buffer 50, pH 7.4, KF 50, NaCl 75, EDTA 2.5, 0.01% NaN₃, and Tris 25, pH 7.4). Both samples for Western blot and PKA phosphorylation were heated in gel-loading buffer (2% SDS, 10% glycerol, 63 mmol/L TRIS, 14 mmol/L 2-mercaptoethanol, and 0.2% bromophenol blue) and analyzed on 8% polyacrylamide gel. The protein from gel was transferred to nitrocellulose membrane. The membrane for in vitro phosphorylation by PKA was exposed to Kodak XOMAT-AR at -80°C with an intensifying screen. Membranes for Western blot were blocked in PBS containing 5% nonfat dry milk. Primary antibody (rabbit NCX antibody, Swant, Switzerland) was incubated at room temperature for 3 hours. After washing, a peroxidase-conjugated secondary antibody was added, and the membrane was incubated for 1 hour. The membrane was washed and developed by peroxidase-catalyzed chemiluminescence of luminol (Amersham). Signals were detected with Hyperfilm-ECL. The band density for each sample in films was digitalized by software (NIH image) and corrected for protein loading.

Electrophysiology
Whole-cell recordings were obtained at 37°C using standard patch-clamp techniques. Membrane current was assessed by use of an Axopatch-100A amplifier and a 1/100 CV-3 headstage (Axon Instruments). Experimental control, data acquisition, and data analysis were accomplished by use of the software package Pclamp 8.0 with the Digidata 1200 acquisition system (Axon Instruments). Patch pipettes were pulled from thin-walled glass capillary tubes and heat-polished. The electrode resistance ranged from 1 to 2 M. The external solution contained (in mmol/L) NaCl 145, MgCl₂ 1, HEPES 5, CaCl₂ 2, CsCl 5, and glucose 10 (pH 7.4, adjusted with NaOH). Ouabain (0.02 mmol/L) and nifedipine (0.01 mmol/L) were added to the solution. The effects of full-scale ß-adrenergic stimulation were achieved by the addition of isoproterenol (ISO, 2 µmol/L), forskolin (5 µmol/L), 8-Br-cAMP (cAMP, 1 mmol/L), or okadaic acid (OKA, 1 µmol/L). The internal solution contained (in mmol/L) CsCl 65, NaCl 20, Na₂ATP 5, CaCl₂ 6, MgCl₂ 4, HEPES 10, tetraethyl ammonium chloride (TEA) 20, and EGTA 21 and ryanodine 0.5 µmol/L (pH 7.2, adjusted with CsOH). In a separate set of experiments, protein phosphatase type 1 (PP1, 10 U/mL) was added to the internal solution to explore the role of tonic phosphorylation of the NCX in the increased basal Ni²⁺-sensitive NCX current (I_NCX) in HF. Membrane currents were elicited by using standard voltage ramp protocol. From a holding potential of -40 mV, a 100-ms step depolarization to +80 mV was followed by a descending voltage ramp (from +80 mV to -120 mV at 100 mV/sec). The protocol was applied every 10 seconds. I_NCX was measured as the Ni²⁺-sensitive current. Ni²⁺ (5 mmol/L) was added to define the fraction of current that derives from NCX (total current remaining after subtraction of post-Ni²⁺ trace). Membrane capacitance was directly read from the membrane test function of Pclamp 8.0 before compensating series resistance and membrane capacitance. The mean capacity of pig ventricular cells was 155±6 pF in failing cells (n=70 versus 78±2 pF in control cells, n=94, P<0.01), consistent with myocyte hypertrophy. The calcium-activated chloride current was not blocked. However, in a separate group of experiments using an identical protocol in 11 cells, Ni²⁺-sensitive currents were measured before and after the addition of 100 µmol/L niflumic acid (Sigma). We found no evidence of calcium-activated chloride current in this species, similar to reports in guinea pigs.

Data Statistical Analysis
Data are presented as the mean±SEM. Significant differences were evaluated by ANOVA or paired or unpaired t test, as appropriate. P<0.05 was regarded as a statistically significant finding.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Heart Failure Increases NCX Expression and Activity
Using a specific polyclonal antibody directed against canine NCX protein, we investigated the NCX expression in both control and failing myocardium. Figure 1 is a representative Western blot showing an increase of NCX protein in failing myocardium (120 kDa, the full-length exchanger, and 80 kDa, a putative proteolytic fragment of the exchanger) when normalized to total protein for each sample. Mean data from 6 controls and 6 failing animals confirmed a significant elevation of NCX protein in failing myocardium compared with control myocardium (P<0.05, Figure 1B). These data are comparable with those reported in pacing-induced failing canine and rabbit models.^4,5 To determine whether the increased protein level of NCX is correlated with altered NCX activity, we measured I_NCX using a voltage ramp protocol. Figure 1C represents I_NCX normalized to cell capacitance from control and failing myocytes, which showed that both outward and inward I_NCX were significantly enhanced in failing myocytes compared with control myocytes. The apparent reversal potential was unchanged in the two groups. Figure 1D is the mean data of the current-voltage relationship of I_NCX in control and failing myocytes. It is clear that HF increased NCX expression and its activity in pig ventricular myocytes, but the increase in I_NCX is much greater than one would predict from the smaller increase in protein. These results suggest that the increase in basal NCX activity seen in HF is not merely attributable to elevated NCX protein. Other means of increasing NCX activity, such as Ca²⁺-dependent activation or PKA phosphorylation, need to be considered. Given that [Ca²⁺]_i was buffered in these experiments, we focused on the role of PKA phosphorylation in modulating I_NCX in HF.

View larger version (39K): [in this window] [in a new window]   Figure 1. Western blot analysis of NCX protein and INCX elicited by voltage ramp in control or failing myocytes. A, Typical Western blot showing an increase in NCX protein in failing myocytes compared with control at 120- and 80-kDa bands from the same amount of total myocyte protein. B, Density of Western blot NCX bands from control or failing myocytes averaged by the number of animals (*P<0.05 failing vs control). C, Representative INCX normalized to cell capacitance from unstimulated control (gray line) and failing (black line) myocytes, showing significantly enhanced outward and inward INCX in failing myocytes compared with control. D, Averaged current-voltage relationship of INCX in myocytes from representative control (open symbols) or failing (filled symbols) pig hearts.

ß-Adrenergic Responsiveness of NCX Current in Heart Failure
Circulating norepinephrine levels are markedly increased in heart failure,¹³ and this could activate NCX activity via cAMP-dependent PKA modulation. In this study we investigated ß-AR responsiveness of NCX in normal and failing myocytes. Figure 2A1 shows a representative trace of I_NCX from control myocytes in basal conditions or in the presence of ISO. ISO markedly increased outward and inward I_NCX in control myocytes. The mean current-voltage relationship (Figure 2A2) reveals that ISO significantly increased I_NCX without any significant shift in the reversal potential. Figure 2A3 represents the percentage increase in the peak current density (at either +80 mV or -120 mV) before and after application of ISO. In control cells, ISO increased I_NCX by nearly 500%; however, in failing myocytes, ISO only enhanced the peak I_NCX by 100% (Figure 2B3). Moreover, the maximum current stimulated by ISO in failing myocytes is only equal to that seen in control myocytes (Figure 2B2), despite the fact that failing myocytes expressed increased NCX protein. Previous studies established that reduced ß-adrenergic receptor number and downregulated adenylyl cyclase activity were found in HF,¹⁴ which might account for the blunted ß-adrenergic responsiveness of NCX current in HF. To test this hypothesis, we additionally exposed failing cells to either forskolin, an adenylyl cyclase activator, or 8-Br-cAMP, which directly stimulates PKA. The results showed that the percentage increase in the peak currents was similar in magnitude compared with the ISO-induced currents (Figure 3, P=0.74, ANOVA). These results suggest that the blunted effect of NCX received in HF was not secondary to decreased ß-adrenergic receptors or downregulated adenylyl cyclase activity in HF.

View larger version (26K): [in this window] [in a new window]   Figure 2. NCX responsiveness to ISO in control and failing myocytes. A1, Representative INCX from control myocytes in the presence (black) or absence (gray) of ISO (2 µmol/L), showing ISO markedly increased outward and inward INCX. A2, Average current-voltage relationship confirming ISO increased outward and inward INCX without changes of reversal potential. A3, Mean increase of the peak outward and inward INCX induced by ISO [(IISO-Ibasal)/Ibasalx100] revealing ISO increased INCX by 500% in control myocytes (P<0.01, ISO vs basal, n=18). B, INCX from failing myocytes in presence (black) or absence (gray) of ISO. Format analogous to that in panel A, but ISO only increased the peak INCX by 100% (P<0.01, ISO vs basal, n=37), which is significantly less than in control myocytes (P<0.05, failure vs control).

View larger version (22K): [in this window] [in a new window]   Figure 3. Comparison of the increase in INCX induced by ß-adrenergic agonists in failing myocytes. The increase of the mean peak INCX induced by ISO (2 µmol/L, n=37), forskolin (FOR, 5 µmol/L, n=7) or 8-Br-cAMP (cAMP, 1 mmol/L, n=10); there were no significant differences among the different agonists (P>0.05, ANOVA).

PKA Hyperphosphorylation of NCX Protein in HF
One possible explanation for our findings of increased basal NCX activity combined with blunted ß-AR effect in HF would be the presence of persistent NCX protein phosphorylation. Two groups have reported increased tonic basal phosphorylation (hyperphosphorylation) of calcium-handling proteins in both failing human heart and the canine model of HF, ie, the L-type Ca²⁺ channel and RyR.^15,16 To test whether the NCX protein undergoes hyperphosphorylation in a similar manner, we examined specific PKA phosphorylation of the NCX in control and failing hearts using back-phosphorylation with PKA. This approach was chosen because of the difficulties inherent in making direct measurements of phosphorylation levels in normal and heart failure cells. In vivo labeling of cells with ³²P0₄ is very inefficient, and few counts can be detected in target protein. This is compounded by the problem that it is difficult to obtain primary cells in sufficient numbers for biochemical analysis. Others investigators have tried without success to detect phosphorylation in cultured cells overexpressing NCX1, even though an enhancement in NCX1 activity can be detected.³⁰ It may be that serine/threonine phosphatases may act to dephosphorylate NCX1 while the protein is extracted. These concerns make this approach less likely to yield positive results when compared with the back-phosphorylation approach undertaken in the present study.

The NCX proteins were precipitated by NCX antibody from control and failing heart tissues and then equally divided into 2 portions. One was analyzed by Western blot, and the other was in vitro phosphorylated by PKA-CS and [³²P] ATP. Figure 4A is a representative autoradiograph for a control and 2 failing heart samples, which shows that NCX protein in failing heart is markedly less phosphorylated by PKA in vitro compared with control heart despite the presence of a higher level of NCX protein indicated by Western blot. After normalization for the amount of NCX protein (Western blot) from 5 control and 5 failing hearts, analysis confirmed that in control hearts the amount of phosphorylation induced by PKA in vitro was twice that in failing heart (Figure 4B). This result suggests that NCX protein was hyperphosphorylated in HF.

View larger version (27K): [in this window] [in a new window]   Figure 4. NCX protein is phosphorylated by PKA in vitro. A, Representative autoradiograph (top) of NCX protein at 120 kDa phosphorylated by PKA-CS and [32P] ATP in vitro in control (C) or failing myocardium (F1 and F2) and the total amount of NCX protein indicated by Western blot (bottom) in above reaction, showing that NCX protein in failing myocardium was markedly less phosphorylated by PKA in vitro compared with control despite the presence of a higher level of protein in HF. B, Mean density of autoradiograph normalized to amount of NCX protein in panel A from 5 control and 5 failing animals (*P<0.05, failing vs control). These data suggest that the NCX protein from failing animals has a reduced phosphorylation capacity consistent with hyperphosphorylation in vivo.

Protein Phosphatase and NCX Phosphorylation in HF
Alterations in the basal phosphorylation state of NCX are likely to reflect changes in the balance of activities of PKA and protein phosphatases. Increased NCX phosphorylation in HF, therefore, could result either from increased PKA activity or decreased phosphatase activity. To examine the effect of protein phosphatase on the NCX in control and failing myocytes, we measured I_NCX in the presence of OKA, a protein phosphatase inhibitor. Figure 5A1 shows a representative trace of NCX current from control myocytes in the basal state or after stimulation with OKA. OKA significantly increased I_NCX to a similar level as seen after ISO (Figures 5A2 and 5A3). Again, in failing cells, the addition of OKA only results in a small increase in peak current (Figures 5B2 and 5B3). These data indicate that endogenous tonic PKA activity is present in control myocytes and that some residual phosphatase activity is present in our failing cells as well. Finally, PP1 (10 U/mL) was dialyzed into the intracellular solution of control and failing myocytes, and peak currents were compared with untreated cells from the same animal. Dialysis of PP1 resulted in a 73% reduction in mean peak current (Figures 6B1, 6B2, and 6B3, P<0.05) in failing myocytes but only reduced the basal current by 20% (P=0.3) in control myocytes (Figures 6A1, 6A2, and 6A3). These data additionally support the hypothesis that NCX is hyperphosphorylated in HF. The fact that increasing phosphatase activity results in normalization of the NCX current is consistent with a reduction in local protein phosphatase activity in heart failure.

View larger version (27K): [in this window] [in a new window]   Figure 5. Effects of the nonspecific PP1 inhibitor, OKA, on INCX in control or failing myocytes. A1, Representative INCX from control myocytes in the presence (black) or absence (gray) of OKA (1 µmol/L), showing OKA markedly increased outward and inward INCX. A2, Average current-voltage relationship confirming OKA increased outward and inward INCX without shifting the reversal potential. A3, Percentage increase in peak INCX induced by OKA [(IOKA-Ibasal)/Ibasalx100]; OKA increased INCX >500% (*P<0.01, OKA vs basal, n=8). B. INCX from failing myocytes in presence (black) or absence (gray) of OKA. Format analogous to that in panel A, but OKA only increased the peak INCX by 200% (*P<0.01, OKA vs basal, n=11), which is significantly smaller than that in control myocytes (P<0.05 failure+OKA vs control+OKA).

View larger version (26K): [in this window] [in a new window]   Figure 6. Effects of PP1 on INCX in control or failing myocytes. A1, Representative INCX from control myocytes in the presence (black) or absence (gray) of PP1 (10 U/mL, dialyzed form internal solution); PP1 has no effect on outward and inward INCX. A2, Mean current-voltage relationship confirming that PP1 has no significant effects on INCX and reversal potential. A3, Mean peak outward (top, at +80 mV) and inward (bottom, at -120 mV) current density in the presence (black) or absence (white) of PP1 in control myocytes (P=0.3, PP1, n=9, versus no PP1, n=8). B, INCX from failing myocytes in presence (black) or absence (white) of PP1. Format analogous to that in panel A, but PP1 significantly reduced the peak INCX in failing cells (*P<0.01, PP1, n=9, vs no PP1, n=11).

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
In this study, we investigated NCX expression and activity under basal conditions and after ß-AR stimulation in control and failing hearts. We demonstrated that HF modestly increased NCX protein level and activity in basal conditions but decreased NCX ß-AR responsiveness and that this alteration is not attributable to downregulation of the ß-AR pathway or decreased ß-AR receptor number but attributable to hyperphosphorylation of the NCX in HF. We additionally show that the hyperphosphorylation of the protein could be attributable to reduced protein phosphatase activity in HF. This information yields insight into NCX regulation in HF that may be helpful in directing future therapeutic strategies aimed at altering NCX activity in heart failure.

HF Increases NCX Expression and Activity
Several groups have reported that HF increases NCX expression or function in failing human heart and animal models of HF. However, there are also discrepant studies reporting no change or a decrease in NCX.^19,20 Some have suggested that the increase in NCX activity is not necessarily associated with increased NCX expression (eg, in the absence of changes in expression level, function could still be elevated).²¹ One group has reported significant heterogeneity in NCX levels among patients undergoing heart transplantation, correlating an increase in NCX with better diastolic relaxation²² but higher-grade arrhythmias. Intriguingly, plasma norepinephrine levels correlated with NCX levels, suggesting that increased sympathetic activity may drive NCX expression.²³ In the present study, we found that HF was associated with a modest increase in NCX expression and yet a disproportionately large increase in function attributable to persistent hyperphosphorylation of the protein. Exploring the mechanism controlling the development of hyperphosphorylation may be critical to understanding the role of the NCX in the variability of phenotype and rate of progression manifested by individuals with congestive heart failure.

Blunted NCX ß-AR Responsiveness and PKA Phosphorylation in HF
Reduction in the extent of ß-AR–mediated contractile reserve is well described in HF. Although both downregulation of the ß-AR pathway and decreased number of ß-AR receptors in HF have been reported, recent studies have suggested that the L-type Ca²⁺ channel and RyR are hyperphosphorylated in HF, resulting in increased basal open probability but reduced responsiveness to ß-AR stimulation. Both the L-type Ca²⁺ channel and RyR are crucial components controlling the amplitude of Ca²⁺ transients in excitation-contraction coupling, and their downregulation may partially explain the depressed ß-AR–mediated increase in contractility seen in HF. The role of the NCX in normal and abnormal E-C coupling is far from clear, although recent data suggest a role for reverse-mode exchange (Na⁺ efflux, Ca²⁺ influx) as an important means of enhancing the gain of calcium-induced calcium release after ß-AR stimulation in normal hearts.²⁴ In the present study, we found that HF resulted in decreased NCX responsiveness to ß-AR stimulation attributable to NCX hyperphosphorylation, which may contribute to the reduced contractile reserve seen in this condition. Taken together, these studies indicate that induction of the hyperphosphorylation state is an important and shared feature of the HF phenotype and possibly an underlying mechanism not only for depressed responsiveness but also altered calcium handling in HF. If so, specific therapeutic modalities aimed at preventing or reversing the hyperphosphorylation state may be useful. Marx et al¹⁶ have shown that ß-AR blockade results in a reduction in hyperphosphorylation associated with improved function in dogs, although others have not found a bradycardia-independent effect of ß-AR blockade on systolic function.²⁵ Significant loss of intracellular Mg²⁺ occurs in HF,²⁶ and this is associated with increased NCX current.²⁷ Because both PKA and protein phosphatases are Mg²⁺ dependent, loss of cellular free magnesium could contribute to decreased phosphorylation and dephosphorylation.²⁸

Marx et al¹⁶ have reported that protein phosphatases PP1 and PP2A form a complex with PKA and the RyR that is altered in HF, resulting in a reduced concentration of phosphatases coprecipitating with the RyR despite increased total cellular PP1 content. We found that the increased basal NCX current observed in myocytes from failing animals was significantly reduced by intracellular dialysis with PP1, which is consistent with reduction of protein phosphatase activity as a cause of hyperphosphorylation of this protein as well. It is unknown whether the NCX is also associated with a regulatory complex containing a protein phosphatase or if a decrease in local phosphatase activity occurs in HF. Indeed, even the site at which PKA-mediated hyperphosphorylation of the NCX occurs is not clear. The rat cardiac NCX1 sequence presents 5 probable sites, of which 3 are intracellular (threonine at 74 and 618; serine at 389).³¹ At present, we do not know which site or sites are phosphorylated; this is an ongoing investigation.

Another surprising finding in the present study is the observation that the maximum current stimulated by ISO in failing myocytes is not greater than that in control myocytes (Figure 2B2), despite the fact that failing myocytes express increased NCX protein. This alteration is not attributable to downregulation of the ß-AR pathway or decreased ß-AR receptor number, because forskolin, adenylyl cyclase-activator, and 8-Br-cAMP could not increase this relatively depressed response to ß-AR stimulation. Because the whole-cell current density is determined by the total number of NCX transporters and the transfer rate, the maximum current stimulated by ß-AR agonist in failing myocytes should be much greater than that in control myocytes if NCX protein is overexpressed in HF. One hypothesis to explain the reduced maximal current in HF would be a shift in the expressed NCX isoform. Ruknudin et al²⁹ have reported that the cardiac isoform of NCX is more sensitive to PKA-mediated phosphorylation than the renal isoform. Whether HF results in a partial isoform shift in NCX protein to one that is resistant to phosphorylation is worthy of additional investigation.

Clinical Implications
Fifty percent of the deaths in patients with HF are sudden and presumably arrhythmic. Increased NCX activity in heart failure is likely to result in an increase in depolarizing current,⁷ particularly when associated with spontaneous calcium release from the RyR. These afterdepolarizations are important triggers of arrhythmia in heart failure. The present study shows that PKA-mediated protein hyperphosphorylation underlies increased NCX activity, complementing previous reports of increased conductance through the RyR and the sarcolemmal calcium channel. Hyperphosphorylation, therefore, represents a tempting therapeutic target for reducing arrhythmia in heart failure.

	Acknowledgments

 
This work was supported in part by grants 9807745-AHA (to M.C.H.), 0265463U-AHA (to S.K.W.), CO83 MD (to S.U.H.), NIH HL62521 (to D.H.S.) and 9730173N-AHA (to A.M.R.).

	Footnotes

 
The views expressed in this article reflect the opinions of the authors only and not the official policy of the Uniformed Services University or the Department of Defense.

Received November 13, 2002; revision received March 18, 2003; accepted March 20, 2003.

	References

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