CLINICAL PERSPECTIVE

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(Circulation. 2007;115:1225-1233.)
© 2007 American Heart Association, Inc.

Heart Failure

Muscarinic Modulation of the Sodium-Calcium Exchanger in Heart Failure

Shao-kui Wei, MD; Abdul M. Ruknudin, PhD; Matie Shou, MD; John M. McCurley, MD; Stephen U. Hanlon, MD; Eric Elgin, MD; Dan H. Schulze, PhD; Mark C.P. Haigney, MD

From the Division of Cardiology, Department of Medicine, Uniformed Services University of the Health Sciences, Bethesda, Md (S.W., M.S., J.M.M., S.U.H., M.C.P.H.); Department of Microbiology and Immunology, University of Maryland, School of Medicine, Baltimore (A.M.R., D.H.S.); Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, Baltimore, Md (A.M.R.); and Division of Cardiology, Walter Reed Army Medical Center, Washington, DC (E.E.).

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

Received July 25, 2006; accepted December 29, 2006.

	Abstract

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Background— The Na-Ca exchanger (NCX) is a critical calcium efflux pathway in excitable cells, but little is known regarding its autonomic regulation.

Methods and Results— We investigated ß-adrenergic receptor and muscarinic receptor regulation of the cardiac NCX in control and heart failure (HF) conditions in atrially paced pigs. NCX current in myocytes from control swine hearts was significantly increased by isoproterenol, and this response was reversed by concurrent muscarinic receptor stimulation with the addition of carbachol, demonstrating "accentuated antagonism." Okadaic acid eliminated the inhibitory effect of carbachol on isoproterenol-stimulated NCX current, indicating that muscarinic receptor regulation operates via protein phosphatase–induced dephosphorylation. However, in myocytes from atrially paced tachycardia-induced HF pigs, the NCX current was significantly larger at baseline but less responsive to isoproterenol compared with controls, whereas carbachol failed to inhibit isoproterenol-stimulated NCX current, and 8-Br-cGMP did not restore muscarinic responsiveness. Protein phosphatase type 1 dialysis significantly reduced NCX current in failing but not control cells, consistent with NCX hyperphosphorylation in HF. Protein phosphatase type 1 levels associated with NCX were significantly depressed in HF pigs compared with control, and total phosphatase activity associated with NCX was significantly decreased.

Conclusions— We conclude that the NCX is autonomically modulated, but HF reduces the level and activity of associated phosphatases; defective dephosphorylation then "locks" the exchanger in a highly active state.

Key Words: calcium • electrophysiology • heart failure • receptors, adrenergic, beta • sodium

	Introduction

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The cardiac Na-Ca exchanger (NCX), a protein found in the sarcoplasmic membrane, acts as the major Ca²⁺ efflux path and is an important Ca²⁺ handling protein regulating intracellular Ca²⁺ in excitation-contraction coupling in the heart. The NCX plays an important role in pathological states as well, acting as a major Ca²⁺ entry site during ischemia/reperfusion.¹ In heart failure (HF), the NCX is significantly upregulated in both human² and animal models,^3,4 which may significantly impair cardiac contractility by reducing sarcoplasmic reticular Ca²⁺ content via premature Ca²⁺ efflux.⁵ It may also promote unstable repolarization with early and/or delayed afterdepolarizations, triggering fatal ventricular arrhythmias.⁶

Clinical Perspective p 1233

Cardiac output is controlled on a beat-to-beat basis by the interaction of the sympathetic and parasympathetic nervous systems. Cardiac sympathetic stimulation activates the ß-adrenergic receptor (ß-AR), whereas parasympathetic stimulation acts on cardiac muscarinic receptor (M-2) systems. These signaling systems interact to regulate cardiac contractility and rate by modulating critical effector proteins⁷ via "accentuated antagonism,"⁸ a phenomenon in which the effect of sympathetic stimulation is rapidly reversed by parasympathetic stimulation despite continued application of the initial stimulus. Accentuated antagonism allows rapid increases in cardiac output in response to physiological challenge while preventing toxicity from excess adrenergic tone.⁹ In HF both ß-AR and M-2 cardiac responsiveness are depressed, contributing to lost cardiac reserve, increased arrhythmic susceptibility,¹⁰ and death. However, the underlying cellular mechanism of these phenomena is far from clear. Some studies have attributed this loss of autonomic responsiveness to the desensitization of ß-AR receptors and/or downregulation of signal transduction in HF.^11,12 However, recent work suggests that the L-type Ca²⁺ channel and ryanodine receptor are tonically phosphorylated ("hyperphosphorylated") at baseline in failing human myocytes,^13–16 which could contribute to depressed ß-AR responsiveness in cardiac function in HF. Some investigators, but not all, have suggested that the NCX is regulated by ß-AR^17–19 and M-2 systems.²⁰ Schulze et al²¹ reported that the NCX in heart is found in a macromolecular complex containing protein kinase A (PKA), protein kinase C, and the protein phosphatases PP1 and PP2a. We recently reported that the NCX current in HF was increased in the basal state,²² manifesting blunted ß-AR regulation, and that these alterations are due to NCX hyperphosphorylation in HF.²³

The effect of hyperphosphorylation on the muscarinic modulation of the affected proteins has not been investigated previously. In the present study, we investigated ß-AR and M-2 regulation of the NCX in both control and HF states using a pacing-induced failing pig model. Our new model is similar to that reported previously except that the present study was performed in atrially (as opposed to ventricularly) paced animals. Rapid atrial pacing results in ventricular dilatation, neurohormonal activation, and loss of ß-AR responsiveness similar to other HF models.^24,25 The change in protocol was introduced for the following reasons: ventricular pacing bypasses the His-Purkinje conduction system and alters the normal pattern of ventricular depolarization, whereas atrial pacing does not (in the absence of conduction system disease).²⁶ Altered ventricular depolarization has numerous effects on cellular protein expression, including the distribution of gap junctions²⁷ and NCX protein density (David Rosenbaum, MD, PhD, personal communication, June 6, 2005). Ventricular pacing per se may introduce phenotypic changes in ventricular myocytes that confound our ability to assess the effect of HF. Additionally, ventricular pacing causes significant changes in ventricular repolarization, preventing a meaningful assessment of the impact of HF on the surface ECG and T wave. Using our refined HF model, we found that in HF the NCX was highly active and displayed markedly depressed ß-AR and M-2 responsiveness. The common pathway of these alterations is downregulation of protein phosphatase expression and activity associated with the NCX, resulting in defective NCX dephosphorylation in HF.

	Methods

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Pacing-Induced Pig HF Model
Induction of HF and ventricular cardiomyocyte isolation and culture were performed as described previously,^22,23 with the use of protocols approved by the university’s Institutional Animal Care and Use Committee, except that the animals were atrially rather than ventricularly paced. The investigation conforms to the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85-23, revised 1996). In brief, Yorkshire pigs (Animal Biotech Industries, Danboro, Pa) of either sex 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. An active-fixation pacing lead was advanced under fluoroscopy to the right atrial appendage. Adequate positioning was confirmed by pacing threshold and P-wave sensing, and the lead was sutured to underlying fascia. After 24 to 48 hours of recovery, pacing was initiated at 200 bpm. All animals underwent transthoracic echocardiography every 5 days. The primary end point of significant left ventricular dysfunction was defined prospectively as a fractional shortening <16% or 2 SDs below the mean fractional shortening in historical controls (32±8%; n=27). Animals were paced until their fractional shortening fell below 16%, at which point they were euthanized.

Cardiomyocyte Isolation and Culture
On development of severe 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 with the same solution but containing 0.24% (wt/vol) collagenase type I (Sigma, St Louis, Mo) and 0.028% protease XIV (Sigma) for 12 minutes 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 excised, 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-shaped 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 µg/mL penicillin, 100 units/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 phenotype with regard to NCX function and ß-AR regulation.

Electrophysiology
Whole-cell recordings were obtained at 37°C with the use of standard patch-clamp techniques. Membrane current was assessed by use of an Axopatch-100A amplifier and a 1/100 CV-3 head stage (Axon Instruments). Experimental control, data acquisition, and data analysis were accomplished with the 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 the following (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 full scale of ß-adrenergic stimulation was achieved by addition of isoproterenol (2 µmol/L). The internal solution contained the following (in mmol/L): CsCl 65, NaCl 20, Na₂ATP 5, CaCl₂ 6, MgCl₂ 4, HEPES 10, tetraethyl ammonium chloride 20, EGTA 21, and ryanodine 0.05 (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 I_NCX in HF. Membrane currents were elicited with the use of 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/s). The protocol was applied every 10 seconds. I_NCX was measured as the Ni-sensitive current. Ni²⁺ (5 mmol/L) was added to define the fraction of current that derives from NCX (the difference between total current and post-Ni²⁺ current). Membrane capacitance was read directly from the membrane test of Pclamp 8.0 before compensating for series resistance and membrane capacitance.

Identity of the Bidirectional, Ni-Sensitive Current
To test whether our recordings were significantly contaminated by the cAMP-dependent Cl^– current (CFTR) or the Ca-activated Cl^– current, we applied niflumic acid (100 µmol/L, Ca-activated Cl^– blocker) and glibenclamide (100 µmol/L, CFTR blocker). In separate experiments, we tested whether reducing extracellular Cl^– (from 145 to 8 mmol/L) and whether removing extracellular Ca²⁺ would rapidly and reversibly suppress the isoproterenol-stimulated current.

Detection and Quantification of the Phosphatases
The total protein from ventricles of normal and failure pig hearts was extracted, and the Western blots were prepared as previously described.^21,23 The NCX antigen complex NCX antibody/Protein A Sepharose was obtained and prepared for Western blots and then immunoblotted with PP2a antibody (BD Biosciences, San Jose, Calif). After the PP2a in the immunoprecipitated NCX complex was identified, the same nitrocellulose membrane was reprobed with NCX antibody after the membrane was stripped of antibodies from the previous experiment. Similarly, another Western blot membrane was immunoblotted with PP1 antibody (Upstate Cell Signaling Solutions, Charlottesville, Va) and then with NCX antibody. With the use of ECL systems (GE Healthcare, Lake Placid, NY), the results were recorded on Biomax Kodak film. The images in the negative films were scanned and analyzed with the use of ImageQuant software (GE Healthcare Systems). Additionally, the ß-1, ß-2, and muscarinic receptor proteins were immunoprecipitated with the use of the appropriate antibody and quantified by Western blot.

Phosphatase Assay
To analyze the total activity of the phosphatases in heart extract and the phosphatases associated with the NCX complex, para-nitrophenyl phosphate was used as a substrate, and the measurements were made following the protocol of the Phosphatase Assay Kit (Upstate Cell Signaling Solutions, Lake Placid, NY). Phosphatase activity for the normal and failure heart extracts (each containing 35 µg of total protein) was estimated. The phosphatase activity associated with NCX was determined for proteins binding to NCX. The NCX macromolecular complex was obtained by incubating 1.2 µL of antibody serum (11-13) with the concentrated heart extract of 350 µL overnight. The NCX complex was washed thoroughly in 800 mL of the wash buffer.²⁸ Enzyme activity was measured for 10 minutes at 37°C. The absorbance was measured at 405 nm. The phosphatase activity in normal heart was considered to be 100% and was compared with change in HF activity.

Protein Phosphatase Activity of PP1 and PP2a
The phosphatase activity of normal and failure heart extracts was determined with the use of the specific phosphopeptide (KRpTIRR) as a substrate for PP1 and PP2A phosphatase enzymes. The phosphate released was visualized with the use of malachite green following the manufacturer’s instructions (Serine/Threonine Phosphatase Assay Kit, Upstate Cell Signaling Solutions, Charlottesville, Va). The solutions were incubated for 20 minutes at room temperature, and color development was measured at 620 nm. The amount of enzyme activity was compared with those measured by the para-nitrophenyl phosphate method.

Statistical Analysis
Data are presented as mean±SEM. Continuous variables were compared by paired or unpaired t test. A probability value of <0.05 was regarded as a statistically significant finding.

The authors had full access to and take full responsibility for the integrity of the data. All authors have read and agree to the manuscript as written.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Identity of the Bidirectional, Ni-Sensitive Current
During the voltage clamp protocol, the membrane potential of the cells was initially held at –40 mV to inactivate sodium channels. Cells were then depolarized to 80 mV to induce an outward current, as seen in Figure 1A, which reflects 3 Na⁺ flowing outward for 1 Ca²⁺ inward. The current becomes inward as the cell is hyperpolarized to –120 mV. The protocol is repeated in the presence of Ni²⁺, which blocks the NCX current, and this Ni-insensitive current is subtracted from the initial tracing to yield the Ni-sensitive bidirectional current. The protocol is then repeated in the presence of isoproterenol. If the induced current is indeed due to NCX activity, it should not be sensitive to blockers of Cl^– channels or to the concentration of Cl^– in the bath and pipette but should require the presence of Ca²⁺ in the bath.

View larger version (28K): [in this window] [in a new window]   Figure 1. The identity of the Ni-sensitive, bidirectional current is NCX (INCX), not Ca-activated chloride current and/or cAMP-dependent CFTR current. A, Depiction of the ramp protocol used in the experiments to elicit the current. An example of a raw current tracing is shown in red, then the ramp is repeated in the presence of 5 mmol/L Ni2+ (blue tracing). The green tracing represents the Ni-sensitive component, the difference between the initial tracing and the post-Ni tracing, and is the putative INCX current. B, From left to right, the average peak outward current density (at +70 mV) elicited in the basal state, in the presence of isoproterenol (ISO) (2 µmol/L), and in the presence of isoproterenol with niflumic acid (NIF) (100 µmol/L, blocker of Ca-activated chloride current) and glibenclamide (GLI) (100 µmol/L, blocker of cAMP-sensitive CFTR current). Niflumic acid and glibenclamide had no effect on the current elicited in the presence of isoproterenol (P=0.96; n=4), consistent with absence of significant Ca-activated chloride or CFTR currents contaminating the NCX current after exposure to isoproterenol. C, Representative current tracing of INCX after lowering external Cl– from 145 to 8 mmol/L. Lowering Cl– had no effect on the isoproterenol+ current (left). The mean peak outward current density (at +70 mV) in the high or low [Cl–]o is unchanged (right; P=0.98; n=9). D, Representative current tracing of INCX after removal of Ca2+. Ca2+ removal rapidly and reversibly suppressed the isoproterenol-induced current (left). The mean peak outward current density in the presence or absence of [Ca2+]o (right; *P<0.05; n=7). These results are inconsistent with CFTR current and support the identity of the current as INCX.

Glibenclamide and niflumic acid, blockers of the CFTR and Ca-activated Cl^– currents, respectively, had no effect on the magnitude of the isoproterenol-stimulated, Ni-sensitive current whether given together (Figure 1B) or separately (not shown). In separate experiments, we found that reducing extracellular chloride (from 145 to 8 mmol/L) had no effect on the magnitude of the isoproterenol-stimulated current (Figure 1C), whereas removing Ca²⁺ rapidly and reversibly suppressed the isoproterenol-stimulated current (Figure 1D). Taken together, these data exclude significant contamination by either the CFTR or Ca-activated chloride current and support the identity of the current as NCX.

Blunted ß-AR and M-2 Regulation of the NCX Current in HF
Recent evidence suggests that ß-AR stimulation increases NCX activity in mammalian myocytes and that M-2 stimulation can modulate this effect.^17–20 Figure 2A1 shows a representative tracing of I_NCX from control myocytes in basal conditions, in the presence of the ß-AR agonist isoproterenol (2 µmol/L/L), and in the presence of isoproterenol plus the M-2 agonist carbachol (5 µmol/L). Isoproterenol markedly increased outward and inward I_NCX in control myocytes, and carbachol significantly inhibited the isoproterenol-stimulated effect. The mean population data of peak outward current density (at +70 mV) reveals that isoproterenol increased I_NCX 300% in controls, whereas carbachol almost completely reversed it (P<0.01; Figure 2A2). However, in failing myocytes basal I_NCX was significantly increased compared with control myocytes, whereas isoproterenol manifested blunted stimulation, increasing the peak I_NCX to a significantly lesser extent (Figure 2B2). The application of the M-2 agonist carbachol did not result in a significant inhibitory effect on isoproterenol-stimulated I_NCX in HF cells (Figure 2B2). This is the first direct evidence of blunted M-2 regulation of the NCX current in HF. Our previous study in a ventricularly paced HF model demonstrated that increased basal I_NCX and reduced ß-AR responsiveness are due to hyperphosphorylation of NCX in HF, but the mechanism of hyperphosphorylation and the interaction with the muscarinic system were not elucidated. Possible explanations for the failure of muscarinic modulation in HF include a decrease in M-2 receptor number, downregulated signal transduction, and/or altered protein phosphorylation state. These alternatives are investigated in the following experiments.

View larger version (35K): [in this window] [in a new window]   Figure 2. INCX responsiveness to isoproterenol and carbachol in control and failing myocytes. A1, A representative current tracing of INCX from a control myocyte in the basal state (blue), after exposure to isoproterenol (2 µmol/L, red), and after isoproterenol plus carbachol (5 µmol/L, green). Isoproterenol markedly increased outward and inward INCX, whereas carbachol reversed the increase in INCX induced by isoproterenol, consistent with "accentuated antagonism." A2, The mean data of peak outward current (at +70 mV; the number of independent experiments is shown in parentheses) as described in A1, confirming that isoproterenol (ISO) significantly increased INCX (*P<0.01, isoproterenol vs basal) and that carbachol (CCH) significantly reversed isoproterenol stimulation (#P<0.05, carbachol vs isoproterenol). B, INCX from failing myocytes (format analogous to that in A). B1, In cells from failing animals, basal INCX is significantly increased compared with control myocytes, whereas isoproterenol induced a significantly smaller further increase. Unlike in control cells, carbachol had no effect on the isoproterenol-stimulated current, demonstrating failure of muscarinic-accentuated antagonism. B2, The mean data of peak outward current (at +70 mV; the number of independent experiments is shown in parentheses) in myocytes from failing animals as described in B1, confirming the failure of carbachol to reverse the effect of isoproterenol. *P<0.01.

Altered Receptor Number and Downregulated Signal Transduction Are Excluded for Explaining Blunted ß-AR and M-2 Regulation of NCX Current in HF
We found no evidence of a reduction in either muscarinic or ß-adrenergic receptor protein expression in HF, suggesting that receptor number was not changed drastically in our model and pointing to a defect in signal transduction (see online-only Data Supplement). Stimulation of the cardiac muscarinic receptor (mainly M-2) is thought to activate soluble guanylyl cyclase, leading to an increase in intracellular cyclic GMP (cGMP). This increase in cytosolic cGMP could either activate cGMP-dependent protein kinase G (PKG) to phosphorylate effectors or activate a protein phosphatase to dephosphorylate PKA-induced phosphorylation.

To identify the altered steps resulting in blunted ß-AR and M-2 regulation of I_NCX in HF, we exposed failing cells to 8-Br-cAMP and 8-Br-cGMP to directly stimulate PKA and PKG. In control myocytes, cAMP (1 mmol/L) significantly increased I_NCX in a manner similar to that of isoproterenol, whereas cGMP significantly reversed this effect; cGMP also reversed the effect of isoproterenol (Figure 3). In failing myocytes, however, both cGMP effects were blunted, suggesting that the impaired ß-AR and M-2 regulation is not due to reduced receptor number or uncoupling of the ß-AR and M-2 signaling pathways. Rather, the defect must be downstream from the generation of cAMP and cGMP.

View larger version (40K): [in this window] [in a new window]   Figure 3. INCX responsiveness to isoproterenol and cGMP in control and failing myocytes. Format is analogous to that in Figure 2. In control myocytes, either 8-Br-cAMP (2 mmol/L) or isoproterenol (ISO) (2 µmol/L, applied in separate cells) significantly increased INCX (*P<0.01), and 8-Br-cGMP (2 mmol/L) significantly reversed it (#P<0.05), similar to the effect of carbachol. However, in failing myocytes agonist responses were blunted to both 8-Br-cAMP and isoproterenol, and 8-Br-cGMP failed to significantly reduce the INCX, showing that the failure of muscarinic effect is not due to depression of cGMP but is due instead to a downstream defect.

Muscarinic System Antagonizes ß-AR Stimulation via Protein Phosphatase
Figure 4A shows that okadaic acid (1 µmol/L) significantly reversed the carbachol inhibition of isoproterenol-stimulated NCX current in control myocytes. This result suggests that a protein phosphatase is a crucial component for M-2 regulation of NCX in myocytes and argues for dephosphorylation as opposed to phosphorylation as a mechanism for M-2 modulation of the NCX. Furthermore, if a protein phosphatase is the mediator of M-2 modulation in the control state, it follows that infusion of exogenous protein phosphatase enzyme should reverse isoproterenol stimulation in HF in a manner similar to that of carbachol in the controls. In a separate experiment, dialysis of PP1 (10 U/mL) through the intracellular solution significantly inhibited I_NCX in basal and isoproterenol-stimulated conditions in failing myocytes but had no significant effect on basal current in control myocytes (Figure 4B). These data suggest that M-2 stimulation inhibits isoproterenol-stimulated I_NCX through activation of a protein phosphatase and that excessive protein phosphorylation in HF results in both increased basal activity and decreased ß-AR responsiveness. A unifying hypothesis explaining these phenomena, as well as the failure of the NCX to respond to M-2 stimulation in HF, would be that the protein phosphatase associated with the NCX is significantly downregulated or inhibited in HF. To test this hypothesis, we measured protein phosphatase levels and activity in protein precipitated with the NCX complex.

View larger version (35K): [in this window] [in a new window]   Figure 4. The muscarinic modulation of NCX activity appears to act via a protein phosphatase. A, A representative current tracing showing that protein phosphatase inhibition by okadaic acid (OKA) (1 µmol/L) eliminates the inhibitory effect of carbachol (CCH) on isoproterenol (ISO)-stimulated INCX in control myocytes (left). The mean peak outward current density in the basal state, in the presence of isoproterenol, isoproterenol plus carbachol, and isoproterenol plus carbachol plus okadaic acid are shown in the right panel (P<0.05, ISO+CCH vs ISO or ISO+CCH vs ISO+CCH+OKA; n=7), indicating that the muscarinic inhibition operates through activating a protein phosphatase. B, The effects of intracellularly applied PP1 (10 U/mL) on INCX in myocytes from control and failing (HF) pigs. PP1 significantly depressed basal INCX (HFBasal) current and after isoproterenol stimulation (HFISO) [*P<0.05, PP1(+) vs PP1(–) exposed NCX current in HF]. PP1 had no significant effect on unstimulated control myocytes, suggesting that the NCX is not phosphorylated significantly at baseline.

Protein Phosphatases Associated With NCX in HF
To test whether the protein phosphatases associated with the NCX are decreased in HF, we examined PP1- and PP2a-associated NCX with a specific antibody directed against PP1 and PP2a in NCX proteins immunoprecipitated by NCX antibody. The NCX proteins were immunoprecipitated by NCX antibody from control and failing heart tissues. The proteins in the NCX complex were separated with the use of PAGE and transferred to nitrocellulose membranes. These membranes were immunoblotted with PP2a antibodies, and the same blot was reprobed with NCX antibody after the membrane was stripped of antibodies from a previous experiment. Figure 5A is a representative Western blot of PP1 and PP2a in control and failing heart samples, showing that PP1 protein is significantly reduced in HF. Surprisingly, PP2a expression appears significantly increased in failing heart muscle compared with control in absolute terms (Figure 5B; P<0.05). However, the total amount of NCX protein was increased in failing hearts by 40% (Figure 5B; P<0.05). After normalization for the amount of NCX protein from control and failing hearts, group analysis confirmed that the mean amount of PP1 in failing hearts was 33% that in control hearts (P<0.01), but PP2a levels were not altered with respect to NCX. To test whether this relative shift in protein expression would alter phosphatase activity, we assessed protein phosphatase activity associated with the NCX and in bulk myocardium with para-nitrophenyl phosphate and malachite green methods. Protein phosphatase activity associated with the NCX was significantly depressed in failing heart compared with control, but no significant difference existed between protein phosphatase activity in bulk myocardium between failure and control states (Figure 6). These results provide direct evidence that protein phosphatase activity associated with the NCX is downregulated in HF.

View larger version (49K): [in this window] [in a new window]   Figure 5. Identification and quantification of protein phosphatases associated with NCX protein. The NCX macromolecular complex was immunoprecipitated from normal and failure heart extracts with the use of NCX antibody and Protein A Sepharose beads. The proteins in the NCX complex were separated with the use of PAGE and transferred to nitrocellulose membranes. These membranes were immunoblotted with PP2a antibodies, and the same blot was reprobed with NCX antibody after the membrane was stripped of antibodies from a previous experiment. A shows PP1 protein (top) in the complex immunoprecipitated by NCX antibody, PP2a (middle), and the NCX protein itself in the complex (bottom) in representative control (C) and HF (F) animals. B, The top panel shows the average amount of PP1 protein (±SEM) associated with the NCX complex from 6 HF and 5 normal animals. There was significant reduction of PP1 enzyme associated with NCX in HF (**P<0.01). There was a slight (but significant, *P<0.05) increase in the PP2a enzyme amount associated with NCX in the HF compared with normal hearts (middle). NCX protein (bottom) measured by Western blot was modestly but significantly increased in HF hearts compared with controls (P<0.05).

View larger version (32K): [in this window] [in a new window]   Figure 6. Protein phosphatase activity in normal and failing hearts. The protein phosphatase activity associated with NCX protein complex and in the heart extract was determined with the use of para-nitrophenyl phosphate and phosphopeptide (KRpTIRR) as substrates. A, The amount of general phosphatase activity was reduced in the NCX complex–associated phosphatases (n=6; *P<0.01), but the activity was not significantly different in the heart extracts in HF compared with control pigs. B, The serine/threonine phosphatase activity associated with the NCX was also significantly reduced in HF animals compared with controls (C).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
This is the first study to investigate the interaction of ß-AR and M-2 regulation of the NCX in HF. The principal findings are as follows: (1) muscarinic receptor stimulation in control myocytes significantly reverses ß-AR stimulation of the NCX via activation of a protein phosphatase; (2) in HF, the NCX is "locked" in a relatively high activity state and is insensitive to both ß-AR and M-2 regulation; (3) the common pathway of these alterations is downregulated protein phosphatase activity resulting in defective NCX dephosphorylation in HF; and (4) the profile of protein phosphatases associated with the NCX is significantly changed in HF with a reduction in PP1 but no change in PP2a. These results extend our previous findings that HF results in hyperphosphorylation of the NCX by providing a mechanism for an increased phosphorylation state, and they further explore the impact of this phenomenon on the autonomic modulation of the exchanger. Furthermore, the use of atrially paced animals addresses the concern that NCX hyperphosphorylation may represent an epiphenomenon of ventricular pacing.

Modulation of the NCX by the ß-AR and Muscarinic Systems
Controversy exists regarding whether the cardiac NCX is modulated by PKA.^29,30 The cAMP-dependent Cl^– current (CFTR) and the Ca-activated Cl^– current are reported to be Ni²⁺ sensitive and have reversal potentials similar to those of NCX.³¹ Lin et al³² have suggested that these conductances could contaminate our recordings of NCX, especially during stimulation of the ß-AR, causing an overestimation of the effect of isoproterenol on the NCX. In the present study, we found no effect of significantly reducing extracellular chloride on the bidirectional, Ni²⁺-sensitive current in pig ventricular myocytes. Additionally, we found that the current was Ca²⁺ dependent, which is consistent with the NCX but not CFTR. Our result is consistent with other reports that although the CFTR conductance is highly represented in small animals, it is nearly or completely absent in large animals such as dogs or humans.³³ Further underscoring the significance of interspecies differences, Ginsburg and Bers³⁴ found no evidence of isoproterenol stimulation of NCX in meticulously controlled experiments in nonfailing rabbits. Although we have no experience with this model, one wonders whether differences in phosphatase activity may account for differences in response to isoproterenol; in such a case, an alternative approach using a phosphatase inhibitor might have yielded evidence of NCX augmentation. Our findings, furthermore, are consistent with those of other groups who found significant increases in NCX current after stimulation with isoproterenol.^17,18,20 The exchanger has been reported to be associated with the regulatory R-1 complex comprising PKA, AKAP, PP1, protein kinase C, and PP2a,²¹ an important piece of evidence suggesting autonomic regulation. Nevertheless, the PKA phosphorylation site on the exchanger has not been identified. The rat cardiac NCX1 sequence presents 5 probable sites, of which 3 are intracellular (threonine at 74 and 618; serine at 389). Methodological differences in the assessment of NCX activity may also contribute to conflicting results. Caffeine superfusion during voltage clamp is a time-honored alternative method of detecting and quantifying the exchange current, but for purposes of studying changes induced by phosphorylation of the exchanger, the ramp method may be superior because it should be less sensitive to changes in sarco/endoplasmic reticular Ca²⁺-ATPase function and sarcoplasmic reticular calcium load.

Further evidence that the NCX is regulated by the autonomic nervous system in a manner that is relevant to human disease is accumulating. Although stimulation of the M-2 receptor by carbachol in the absence of preceding ß-AR stimulation has been shown to induce an indirect increase in the NCX current via subsarcolemmal Na⁺ gain,³⁵ the present study confirms the observations of Zhang et al²⁰ that NCX activity is strongly depressed by muscarinic agonists administered during ß-AR stimulation, but the mechanism of signal transduction was not previously elucidated. Investigating the L-type Ca channel, Jiang et al³⁶ reported that M-2 modulates ß-AR stimulation via PKG-induced L-type channel phosphorylation, whereas Shen and Pappano³⁷ have suggested that the primary response of cGMP is to activate protein phosphatases to dephosphorylate PKA-induced phosphorylation. To examine these putative mechanisms, we reasoned that if the muscarinic effect is modulated by phosphorylation of a separate site on the NCX in the presence of isoproterenol, then application of a nonspecific protein phosphatase inhibitor, ie, okadaic acid, should increase (or at least not inhibit) muscarinic modulation of the current by allowing further accumulation of phosphorylation. Alternatively, if M-2 stimulation results in activation of an NCX-associated protein phosphatase, inhibition of that phosphatase by okadaic acid should reverse the effect of M-2 stimulation and restore the current. We found that okadaic acid indeed reversed the carbachol effect, consistent with activation of a phosphatase as the mechanism of muscarinic modulation of the NCX. Finally, Katanosaka et al³⁸ have reported that the C-terminus of the Aß of calcineurin, a phosphatase mechanistically implicated in cardiac hypertrophy, binds to the cytoplasmic loop of the cardiac NCX in hamsters. Prolonged phenylephrine exposure, which would stimulate the -adrenergic system and act as a model of hypertension, resulted in inhibition of Na⁺_o-dependent Ca²⁺ efflux from isolated myocytes in a manner that was prevented by inhibition by calcineurin. This is consistent with differential phosphatase modulation by distinct pathological signaling mechanisms.

Blunted ß-AR and M-2 Regulation in HF
Recent studies have suggested that elevated tonic phosphorylation (hyperphosphorylation) of calcium handling proteins such as the L-type Ca channel and the ryanodine receptor might contribute to increased basal Ca²⁺ permeability and reduced responsiveness to ß-AR stimulation. In our previous study, we also found that the NCX is hyperphosphorylated in HF, suggesting that downregulated protein phosphatase might be the underlying mechanism. In the present study, we have demonstrated reduced PP1 protein and protein phosphatase activity associated with the NCX, providing a mechanism for NCX hyperphosphorylation in HF. The present study differs somewhat from our previous work in that these animals were atrially paced into HF. This change should avoid artifactual changes in myocyte phenotype due to abnormal depolarization; however, the magnitude of the increase in the basal current due to HF appears to be 25% less than we found previously despite similar increases in NCX expression. The most likely explanation is that rapid ventricular pacing induces a more severe HF phenotype because of the introduction of intraventricular and atrioventricular dyssynchrony that would not be appreciated by echocardiograms performed in sinus rhythm.

In the present study, we found that the M-2 receptor agonist carbachol loses its ability to inhibit ß-AR stimulation on the NCX in HF. In contrast to the decreased numbers of ß-AR receptors found in HF, the number of M-2 receptors is either unchanged or increased in HF.³⁹ We found that application of cGMP fails to recover M-2 inhibition of ß-AR signaling, confirming that the defect of muscarinic regulation is not at the M-2 receptor and/or G_i protein and must be downstream of cGMP. However, dialysis with PP1 significantly depressed both NCX current in basal conditions and after isoproterenol stimulation in HF. Furthermore, the protein phosphatase inhibitor okadaic acid eliminates carbachol inhibition of isoproterenol-stimulated I_NCX, indicating that M-2 regulation of the NCX is dependent on protein phosphatase activity. On the basis of these findings, we suggest that both ß-AR and M-2 dysfunction in HF are due to downregulation of protein phosphatase, principally PP1.

Downregulated Protein Phosphatase Associated With NCX in HF
The effect of HF on protein phosphatase expression and activity is clearly complex. Neumann et al⁴⁰ have reported that cardiac PP1 protein and mRNA levels were increased in HF, resulting in decreased phospholamban phosphorylation and depressed sarcoplasmic reticulum–ATPase function, whereas other groups have found decreased local protein phosphatase expression. Marx et al¹⁴ found that the protein levels of both PP1 and PP2a associated with ryanodine receptor were reduced in HF. In the present study, we found that the NCX associates with protein phosphatases in a macromolecular complex in pig hearts, similar to findings in the rat. In contrast to the results of Marx et al (with respect to the ryanodine receptor), however, we found that PP1 expression is decreased, whereas PP2a associated with the NCX is unchanged in HF. Despite this shift in phosphatase profile associated with the complex, we found that total protein phosphatase activity associated with NCX is significantly depressed in HF compared with control (whereas total myocardial activity is unchanged). These results suggest that PP1 might play a principal role in NCX dephosphorylation in the control state, whereas PP2a is predominant in HF. Although it is generally believed that PP1 and PP2a have similar activity in terms of dephosphorylation of serine/threonine sites, the structure, activity, and regulation of these isoforms are distinct.^41,42 PP1 activity is modulated by the inhibitor protein I-1, which is itself activated by PKA phosphorylation. PP2a can dephosphorylate I-1,⁴³ but little is known about the regulation of PP2a itself. A recent report has suggested that the enzyme is inhibited by elevated [Ca²⁺]_I,⁴⁴ whereas overexpression of PP2a has been shown to result in reduce cardiac contractility.⁴⁵ The present study suggests that PP2a activity is not modulated by muscarinic stimulation, resulting in failure of dephosphorylation of the NCX in response to M-2 stimulation. The effect of this shift in phosphatase isoform expression on NCX regulation and the HF phenotype merits further investigation.

Conclusions
Fifty percent of the deaths in patients with HF are sudden and presumably arrhythmic. Increased NCX activity in HF is likely to enhance depolarizing current, particularly when associated with spontaneous calcium release from the sarcoplasmic reticulum, which could result in afterdepolarizations triggering fatal ventricular arrhythmia. Furthermore, excess NCX activity has been tied to depression of the systolic calcium transient in HF, linking the NCX to mechanical pump dysfunction as well. The present study shows that in HF, the NCX is "locked" in a relatively high activity state and insensitive to both ß-AR and M-2 regulation, which could reduce cardiac contractile reserve and increase susceptibility to arrhythmia. The common pathway of these alterations is downregulation of associated protein phosphatase activity, resulting in defective NCX dephosphorylation in HF. Dephosphorylation of the NCX represents a possible therapeutic target for reducing arrhythmia and pump dysfunction in HF.

	Acknowledgments

 
Sources of Funding

The present study was supported in part by grants from the Department of Defense (CO83OD to Dr Haigney; C083QF to Dr McCurley), the National Institutes of Health (HL62521 to Dr Schulze), the National Institutes of Aging (AG-020823 to Dr Ruknudin), and the American Heart Association (0265463U to Dr Wei; 9730173N to Dr Ruknudin).

Disclosures

None.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. Kusuoka H, Camilion de Hurtado MC, Marban E. Role of sodium/calcium exchange in the mechanism of myocardial stunning: protective effect of reperfusion with high sodium solution. J Am Coll Cardiol. 1993; 21: 240–248.[Abstract]
   
2. Hasenfuss G, Schillinger W, Lehnart SE. Relationship between Na⁺-Ca²⁺-exchanger protein levels and diastolic function of failing human myocardium. Circulation. 1999; 99: 641–648.[Abstract/Free Full Text]
   
3. O’Rourke B, Kass DA, Tomaselli GF, Kaab S, Tunin R, Marban E. Mechanisms of altered excitation-contraction coupling in canine tachycardia-induced heart failure, I: experimental studies. Circ Res. 1999; 84: 562–570.[Abstract/Free Full Text]
   
4. Pogwizd SM, Qi M, Yuan W, Samarel AM, Bers DM. Upregulation of Na⁺/Ca²⁺ exchanger expression and function in an arrhythmogenic rabbit model of heart failure. Circ Res. 1999; 85: 1009–1019.[Abstract/Free Full Text]
   
5. Hobai IA, Maack C, O’Rourke B. Partial inhibition of sodium/calcium exchange restores cellular calcium handling in canine heart failure. Circ Res. 2004; 95: 292–299.[Abstract/Free Full Text]
   
6. Pogwizd SM, Schlotthauer K, Li L, Yuan W, Bers DM. Arrhythmogenesis and contractile dysfunction in heart failure: roles of sodium-calcium exchange, inward rectifier potassium current, and residual beta-adrenergic responsiveness. Circ Res. 2001; 88: 1159–1167.[Abstract/Free Full Text]
   
7. Takasago T, Imagawa T, Shigekawa M. Phosphorylation of the cardiac ryanodine receptor by cAMP-dependent protein kinase. J Biochem. 1989; 106: 872–877.[Abstract/Free Full Text]
   
8. Levy MN. Sympathetic-parasympathetic interactions in the heart. Circ Res. 1971; 29: 437.[Free Full Text]
   
9. Vanoli E, Schwartz PJ. Sympathetic-parasympathetic interaction and sudden death. Basic Res Cardiol. 1990; 85: 305–321.[Medline] [Order article via Infotrieve]
   
10. Schwartz PJ. The autonomic nervous system and sudden death. Eur Heart J. 1998; 19: F72–F80.[Medline] [Order article via Infotrieve]
    
11. Bristow MR, Ginsburg R, Minobe W, Cubicciotti RS, Sageman WS, Lurie K, Billingham ME, Harrison DC, Stinson EB. Decreased catecholamine sensitivity and beta-adrenergic-receptor density in failing human hearts. N Engl J Med. 1982; 307: 205–211.[Abstract]
    
12. Cohn JN, Levine TB, Olivari MT, Garberg V, Lura D, Francis GS, Simon AB, Rector T. Plasma norepinephrine as a guide to prognosis in patients with chronic congestive heart failure. N Engl J Med. 1984; 311: 819–823.[Abstract]
    
13. Schroder F, Handrock R, Beuckelmann DJ, Hirt S, Hullin R, Priebe L, Schwinger RH, Weil J, Herzig S. Increased availability and open probability of single L-type calcium channels from failing compared with nonfailing human ventricle. Circulation. 1998; 98: 969–976.[Abstract/Free Full Text]
    
14. Marx SO, Reiken S, Hisamatsu Y, Jayaraman T, Burkhoff D, Rosemblit N, Marks AR. PKA phosphorylation dissociates FKBP12.6 from the calcium release channel (ryanodine receptor): defective regulation in failing hearts. Cell. 2000; 101: 365–376.[CrossRef][Medline] [Order article via Infotrieve]
    
15. Chen X, Piacentino V 3rd, Furukawa S, Goldman B, Margulies KB, Houser SR. L-type Ca2+ channel density and regulation are altered in failing human ventricular myocytes and recover after support with mechanical assist devices. Circ Res. 2002; 91: 517–524.[Abstract/Free Full Text]
    
16. Kamp TJ, He JQ. L-type Ca2+ channels gaining respect in heart failure. Circ Res. 2002; 91: 451–453.[Free Full Text]
    
17. Perchenet L, Hinde AK, Patel KC, Hancox JC, Levi AJ. Stimulation of Na/Ca exchange by the beta-adrenergic/protein kinase A pathway in guinea-pig ventricular myocytes at 37 degrees C. Pflugers Arch. 2000; 439: 822–828.[CrossRef][Medline] [Order article via Infotrieve]
    
18. Ruknudin A, He S, Lederer WJ, Schulze DH. Functional differences between cardiac and renal isoforms of the rat Na+-Ca2+ exchanger NCX expressed in Xenopus oocytes. J Physiol. 2000; 529: 599–610.[Abstract/Free Full Text]
    
19. Wei SK, Hanlon SU, Haigney MC. Beta-adrenergic stimulation of pig myocytes with decreased cytosolic free magnesium prolongs the action potential and enhances triggered activity. J Cardiovasc Electrophysiol. 2002; 13: 587–592.[CrossRef][Medline] [Order article via Infotrieve]
    
20. Zhang YH, Hinde AK, Hancox JC. Anti-adrenergic effect of adenosine on Na(+)-Ca(2+) exchange current recorded from guinea-pig ventricular myocytes. Cell Calcium. 2001; 29: 347–358.[CrossRef][Medline] [Order article via Infotrieve]
    
21. Schulze DH, Muqhal M, Lederer WJ, Ruknudin AM. Sodium/calcium exchanger (NCX) macromolecular complex. J Biol Chem. 2003; 278: 28849–28855.[Abstract/Free Full Text]
    
22. Wei SK, Hanlon SU, Haigney MCP. Decreased -adrenergic responsiveness of Na/Ca exchange current in failing pig myocytes. Ann NY Acad Sci. 2002; 976: 472–476.[Free Full Text]
    
23. Wei SK, Ruknudin A, Hanlon SU, McCurley JM, Schulze DH, Haigney MC. Protein kinase A hyperphosphorylation increases basal current but decreases beta-adrenergic responsiveness of the sarcolemmal Na⁺-Ca²⁺ exchanger in failing pig myocytes. Circ Res. 2003; 92: 897–903.[Abstract/Free Full Text]
    
24. Tanaka R, Fulbright BM, Mukherjee R, Burchell SA, Zile MR, Spinale FG. The cellular basis for the blunted response to -adrenergic stimulation in supraventricular tachycardia-induced cardiomyopathy. J Mol Cell Cardiol. 1993; 25: 1215–1233.[CrossRef][Medline] [Order article via Infotrieve]
    
25. Spinale FG, Fulbright BM, Mukherjee R, Tanaka R, Hu J, Crawford FA, Zile MR. Relationship between ventricular and myocyte function with tachycardia-induced cardiomyopathy. Circ Res. 1992; 71: 174–187.[Abstract/Free Full Text]
    
26. Patberg KW, Rosen MR. Molecular determinants of cardiac memory and their regulation. J Mol Cell Cardiol. 2004; 36: 195–204.[CrossRef][Medline] [Order article via Infotrieve]
    
27. Shvilkin A, Danilo P, Wang J, Burkhoff D, Anyukhovsky EP, Sosunov EA, Hara M, Rosen MR. Evolution and resolution of long-term cardiac memory. Circulation. 1998; 97: 1810–1817.[Abstract/Free Full Text]
    
28. Ivanina T, Perets T, Thornhill WB, Levin G, Dascal N, Lotan I. Phosphorylation by protein kinase A of RCK1 K⁺ channels expressed in Xenopus oocytes. Biochemistry. 1994; 33: 8786–8792.[CrossRef][Medline] [Order article via Infotrieve]
    
29. Condrescu M, Gardner JP, Chernaya G, Aceto JF, Kroupis C, Reeves JP. ATP-dependent regulation of sodium-calcium exchange in Chinese hamster ovary cells transfected with the bovine cardiac sodium-calcium exchanger. J Biol Chem. 1995; 270: 9137–9146.[Abstract/Free Full Text]
    
30. Collins A, Somlyo AV, Hilgemann DW. The giant cardiac membrane patch method: stimulation of outward Na(+)-Ca2+ exchange current by MgATP. J Physiol. 1992; 454: 27–57.[Abstract/Free Full Text]
    
31. Xue L, Jo H, Matsuoka S. Ni2+ inhibits both Na-Ca exchange and cAMP-dependent Cl– currents in guinea-pig ventricular cells. Biophys J. 2003; 86: 613a. Abstract.
    
32. Lin X, Jo H, Sakakibara Y, Tambara K, Kim B, Komeda M, Matsuoka S. Beta-adrenergic stimulation does not activate Na+/Ca2+ exchange current in guinea pig, mouse, and rat ventricular myocytes. Am J Physiol. 2006; 290: C601–C608.[CrossRef]
    
33. Du XY, Finley J, Sorota S. Paucity of CFTR current but modest CFTR immunoreactivity in non-diseased human ventricle. Pflugers Arch. 2000; 440: 61–67.[Medline] [Order article via Infotrieve]
    
34. Ginsburg KS, Bers DM. Isoproterenol does not enhance Ca-dependent Na/Ca exchange current in intact rabbit ventricular myocytes. J Mol Cell Cardiol. 2005; 39: 972–981.[CrossRef][Medline] [Order article via Infotrieve]
    
35. Saeki T, Shen JB, Pappano AJ. Carbachol promotes Na+ entry and augments Na/Ca exchange current in guinea pig ventricular myocytes. Am J Physiol. 1997; 273: H1984–H1993.[Medline] [Order article via Infotrieve]
    
36. Jiang LH, Gawler DJ, Hodson N, Milligan CJ, Pearson HA, Porter V, Wray D. Regulation of cloned cardiac L-type calcium channels by cGMP-dependent protein kinase. J Biol Chem. 2000; 275: 6135–6143.[Abstract/Free Full Text]
    
37. Shen JB, Pappano AJ. On the role of phosphatase in regulation of cardiac L-type calcium current by cyclic GMP. J Pharmacol Exp Ther. 2002; 301: 501–506.[Abstract/Free Full Text]
    
38. Katanosaka Y, Iwata Y, Kobayashi Y, Shibisaki F, Wakabayashi S, Shigekawa M. Calcineurin inhibits Na+/Ca2+ exchange in phenylephrine-treated hypertrophic cardiomyocytes. J Biol Chem. 2005; 280: 5764–5772.[Abstract/Free Full Text]
    
39. Le Guludec D, Cohen-Solal A, Delforge J, Delahaye N, Syrota A, Merlet P. Increased myocardial muscarinic receptor density in idiopathic dilated cardiomyopathy: an in vivo PET study. Circulation. 1997; 96: 3416–3422.[Abstract/Free Full Text]
    
40. Neumann J, Eschenhagen T, Jones LR, Linck B, Schmitz W, Scholz H, Zimmermann N. Increased expression of cardiac phosphatases in patients with end-stage heart failure. J Mol Cell Cardiol. 1997; 29: 265–272.[CrossRef][Medline] [Order article via Infotrieve]
    
41. Luss H, Klein-Wiele O, Boknik P, Herzig S, Knapp J, Linck B, Muller FU, Scheld HH, Schmid C, Schmitz W, Neumann J. Regional expression of protein phosphatase type 1 and 2A catalytic subunit isoforms in the human heart. J Mol Cell Cardiol. 2000; 32: 2349–2359.[CrossRef][Medline] [Order article via Infotrieve]
    
42. duBell WH, Lederer WJ, Rogers TB. Dynamic modulation of excitation-contraction coupling by protein phosphatases in rat ventricular myocytes. J Physiol. 1996; 493: 793–800.[Medline] [Order article via Infotrieve]
    
43. Gupta RC, Neumann J, Watanabe AM, Sabbah HN. Inhibition of type 1 protein phosphatase activity by activation of beta-adrenoceptors in ventricular myocardium. Biochem Pharmacol. 2002; 63: 1069–1076.[CrossRef][Medline] [Order article via Infotrieve]
    
44. Palanivel R, Veluthakal R, Kowluru A. Regulation by glucose and calcium of the carboxylmethylation of the catalytic subunit of protein phosphatase 2A in insulin-secreting INS-1 cells. Am J Physiol. 2004; 286: E1032–E1041.
    
45. Gergs U, Boknik P, Buchwalow I, Fabritz L, Matus M, Justus I, Hanske G, Schmitz W, Neumann J. Overexpression of the catalytic subunit of protein phosphatase 2A impairs cardiac function. J Biol Chem. 2004; 279: 40827–40834.[Abstract/Free Full Text]
    

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CLINICAL PERSPECTIVE

The sodium-calcium exchanger is the most important protein for removing calcium from the cytosol of cardiac muscle cells. Although calcium is needed to drive systolic contraction, excess calcium is associated with arrhythmias and cell death. Despite the importance of the sodium-calcium exchanger in maintaining calcium homeostasis, the mechanisms controlling the activity of the exchanger are poorly understood. Sympathetic nervous system stimulation of the sodium-calcium exchanger by ß-adrenergic agonists increases its activity in healthy pigs. In the present study in cardiac myocytes from healthy and heart failure pigs, the effect of agonists of the parasympathetic nervous system was tested and found to reverse the stimulatory effects of isoproterenol or cAMP analogues. In heart failure, however, the exchanger fails to respond to either sympathetic or parasympathetic nervous system activity but instead remains "locked" in a high activity state. This failure of modulation may contribute to increased myocyte calcium loss in heart failure, contributing to poor systolic contractile performance and arrhythmogenesis.

	Footnotes

 
The online-only Data Supplement, consisting of figures, is available with this article at http://circ.ahajournals.org/cgi/content/ full/CIRCULATIONAHA.106.650416/DC1.

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