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(Circulation. 2000;102:692.)
© 2000 American Heart Association, Inc.

Basic Science Reports

G-Protein ß₃-Subunit 825T Allele Is Associated With Enhanced Human Atrial Inward Rectifier Potassium Currents

D. Dobrev, MD; E. Wettwer, PhD; H. M. Himmel, MD; A. Kortner, MD; E. Kuhlisch, DM; S. Schüler, MD, PhD; W. Siffert, MD, PhD; U. Ravens, MD, PhD

From the Department of Pharmacology and Toxicology (D.D., E.W., H.M.H., U.R.), Cardiovascular Centre Dresden (A.K., S.S.), and Department of Medical Informatics and Biometry (E.K.), Medical Faculty, University of Technology, Dresden; and the Department of Pharmacology, Medical Faculty, Essen University, Essen (W.S.), Germany.

Correspondence to Prof Dr med Ursula Ravens, Institut für Pharmakologie und Toxikologie, Karl-Marx-Straße 3, D-01109 Dresden, Germany. E-mail ravens{at}rcs.urz.tu-dresden.de

	Abstract

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Background—A C825T polymorphism was recently identified in the human gene encoding for the ß₃-subunit of heterotrimeric G proteins. The 825T allele is associated with a splice variant of Gß₃ and enhanced signal transduction. We hypothesized that patients carrying the 825T allele exhibit the modified Gß₃ phenotype. The resulting enhancement of signal transduction should be detectable in the Gß-dimer–mediated acetylcholine-stimulated K⁺ current (I_K,ACh).

Methods and Results—Seventy patients undergoing cardiac surgery were genotyped for the C825T polymorphism. In right atrial myocytes from these patients, the inward rectifier K⁺ currents (I_K1, I_K,ACh) were studied with the whole-cell patch-clamp technique. Background current I_K1 was measured with depolarizing ramp pulses and quantified as inward current at -100 mV; mean amplitudes were (pA/pF) 4.98±0.49 (n=30/93 patients/cells) in patients with CC genotype, 4.25±0.36 (n=31/121 patients/cells) with TC, and 7.46±1.14 (n=9/32 patients/cells; P<0.05) with TT. Conversely, mean I_K,ACh, which is maximally activated by carbachol (2 µmol/L), was reduced in patients with TT genotype (pA/pF, 4.30±1.33, n=9/27 patients/cells; P<0.05) compared with the other 2 groups (6.56±0.54, n=30/80 and 6.16±0.45, n=31/117 patients/cells, for CC and TC genotype, respectively). Essentially similar results were obtained with adenosine (1 mmol/L).

Conclusions—We found an association between the Gß₃ 825T allele and amplitude of human atrial I_K1 and I_K,ACh. Increased background current density in TT carriers could shorten action potential duration and may be due to I_K,ACh being constitutively active in this genotype.

Key Words: myocytes • ions • genes • carbachol • adenosine

	Introduction

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Recent studies in immortalized cell lines from hypertensive individuals have demonstrated enhanced signal transduction between receptors and effectors and suggested involvement of pertussis toxin–sensitive G_i proteins.^{1 2} This elevated activity of signal transduction must be genetically determined, because it is maintained during numerous cell passages in culture. In fact, enhanced G_i-protein activity was traced back to a polymorphism in the gene encoding for the ubiquitously expressed ß₃ subunit of heterotrimeric G proteins.³ Exon 10 of this gene may contain a thymine instead of a cytosine (C825T polymorphism). The 825T allele is associated with alternative splicing of exon 9, resulting in a modified Gß₃-subunit that is shorter by 41 amino acids and more active than the full-size Gß₃-subunit.³ Clinically, the 825T allele is associated with essential hypertension.^{3 4 5 6}

In human atrial myocytes, the signal transduction between stimulation of muscarinic M₂ receptors and opening of acetylcholine-activated K⁺ channels (I_K,ACh) requires pertussis toxin–sensitive G_i proteins. Activated G_i proteins release ß-subunit dimers, which activate I_K,ACh by direct binding to this channel.^{7 8} There are numerous isoforms of the -, ß-, and -subunits. At least 6 isoforms for ß- and 12 for -subunits have been described so far,⁹ but the isoform composition of the ß-subunit involved in activation of human atrial I_K,ACh is not known. I_K,ACh is an inwardly rectifying K⁺ current with a voltage dependence similar to that of the inward rectifier current I_K1, from which it cannot be distinguished at the whole-cell current level. Both channels are of clinical significance, because they contribute substantially to the atrial resting potential and determine the shape of cardiac action potentials during the final phase of repolarization.¹⁰ In addition, I_K,ACh is the major effector of vagal stimulation in atrial myocytes.⁸

The aim of our present study was to discover a putative association between the 825T allele and the activity of a signal transduction cascade that involves the ß-subunits of G_i proteins. We hypothesized that carriers of the 825T allele should exhibit enhanced signaling due to expression of the alternatively spliced, more active Gß₃ gene product. Hence, the response of atrial I_K,ACh stimulation with muscarinic-receptor agonists could serve as a sensitive parameter to monitor G_i-protein activity as the phenotype. To this purpose, using the patch-clamp technique, we studied the inward rectifier currents I_K1 and I_K,ACh in human atrial myocytes from patients receiving coronary artery bypass graft surgery.

	Methods

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Study Population
Seventy consecutive patients who underwent open-heart coronary artery bypass graft surgery were included in this study. Each patient gave written informed consent for the study, which was approved by the local ethics committee (No. EK730698). Exclusion criteria were decompensated left ventricular failure, atrial fibrillation, and chronic pulmonary hypertension. One patient in the group with CC and 3 patients in the group with TT genotype had symptomatic aortic stenosis; none had mitral insufficiency. Before surgery, each patient received a standard ECG, echocardiography, and routine laboratory tests for evaluation of atherosclerosis risk factors. These were defined either by specific drug treatment (see Table 1) or by laboratory analysis with the following values: serum cholesterol >5.5 mmol/L and/or triglycerides >1.7 mmol/L and fasting serum glucose level >5.5 mmol/L. Hypertension was diagnosed with systolic blood pressure >140 mm Hg or diastolic pressure >90 mm Hg and/or the need for antihypertensive pharmacotherapy.

View this table: [in this window] [in a new window]   Table 1. Patients’ Medications in Genotype Groups

The left ventricular end-diastolic dimension and size of the left atrium were determined by M-mode echocardiography according to the recommendations of the American Society of Echocardiography.¹¹ The left ventricular ejection fraction was assessed by biplane angiography. Table 1 shows the patients’ medications. Anticoagulants were withheld 7 days before surgery.

Isolation of Myocytes
Adult human right atrial specimens were obtained from patients undergoing cardiac surgery. They were immediately placed into chilled Ca²⁺-free solution (in mmol/L: NaCl 100, KCl 10, KH₂PO₄ 1.2, MgSO₄ 5, taurine 50, MOPS 5, and glucose 20, with pH adjusted to 7.0 with NaOH) supplemented with 2,3-butanedione monoxime (BDM, 30 mmol/L), chopped into small pieces, and washed 3 times for 3 minutes with Ca²⁺-free Tyrode’s solution. At all steps, the solutions were oxygenated with 100% O₂ at 35°C. Tissue pieces were then transferred into Ca²⁺-free Tyrode’s solution containing 254 U/mL collagenase type I (Worthington) and 0.5 mg/mL protease type XXIV (Sigma Chemical Co) and gently stirred for 15 minutes. Then the Ca²⁺ concentration was raised to 0.2 mmol/L, and the tissue was stirred for 30 minutes more. Stirring was continued with Tyrode’s solution (0.2 mmol/L Ca²⁺) containing only collagenase until rod-shaped striated myocytes were seen (35 minutes).¹² The suspension was centrifuged, and myocytes were resuspended and stored until use in Ca²⁺ (0.5 mmol/L)–containing Tyrode’s solution (without BDM) at room temperature. The yield of elongated quiescent myocytes with clear cross-striations was 19.0±1.6% (n=34).

Measurement of Membrane Currents
The single-electrode whole-cell voltage-clamp method was applied with a List EPC-7 amplifier. pCLAMP 5.5 software (Axon Instruments) was used for data acquisition and analysis. The analog data were filtered at 2 kHz through a 10-pole low-pass Bessel filter (Zeitz Instrumente) and digitized at 1 sample/ms with a TL 125 A/D interface (Axon Instruments) for offline analysis. Electrodes were fabricated from filamented borosilicate glass (Hilgenberg Co) with a programmable horizontal puller (DMZ universal puller, Zeitz). When filled with electrode solution (in mmol/L: K-aspartate 100, NaCl 10, KCl 40, Mg-ATP 5.0, EGTA 2.0, GTP-Tris 0.1, and HEPES 10, with pH adjusted to 7.4 with KOH), the microelectrodes had tip resistances of 2 to 3 M. Seal resistances were usually between 5 and 8 G.

Membrane capacitance (C_M) was routinely measured with depolarizing ramps (1 V/s) from -40 to -35 mV.¹² C_M was compensated up to 100 pF, and series resistance compensation was set to 70%. The liquid junction potential between electrode and standard bath solution was calculated with the software JPCalc, version 2.2, by Barry¹³ and amounted to -12 mV. The mean resting potential was not different between the 3 genotype groups and was -28±1 mV (n=63). Taking into account the calculated liquid junction potential, the resting membrane potential of our cells (-40 mV) was close to the potassium equilibrium potential of -49 mV as calculated with the Nernst equation. The data presented are not corrected for the calculated junction potential.

Because atrial cells have a high input resistance,¹² leak currents may contribute to whole-cell currents. Therefore, ramp currents were corrected by a linear conductance determined at the reversal potential of I_K,ACh after stimulation with carbachol. Mean leak conductance of all cells studied was 670 pS and corresponded well with the residual current obtained in the presence of 1 mmol/L Ba²⁺.

The holding potential was -80 mV. The pulse protocol consisted of a 50-ms step to -100 mV, followed by a depolarizing ramp to -10 mV (800 ms), a 100-ms step to -50 mV, and back to the holding potential. The pulse protocol was elicited at a rate of 0.5 Hz. I_K1 was quantified as either inward current at -100 mV or outward current at -10 mV corrected for cell capacitance (in pA/pF). Once current traces had stabilized, I_K,ACh was elicited by applying either carbachol or adenosine to the bath solution (composition in mmol/L: NaCl 120, KCl 20, MgCl₂ 1, CaCl₂ 2.0, glucose 10, and HEPES 10, with pH adjusted to 7.4 with NaOH). All experiments were carried out at room temperature (22°C to 24°C).

Molecular Analysis of the Gß₃ Gene
Genomic DNA was extracted by standard methods from 2 mL EDTA-anticoagulated blood taken from all patients on the day of surgery. Genotyping was conducted exactly as described previously³ in a blinded manner, ie, the genotyping laboratory was unaware of any patient or electrophysiological data.

Statistical Analysis
For quantitative comparisons, average values were calculated from multiple data obtained from 1 patient. Because the number of cells investigated varied widely in each patient (unbalanced observations), we calculated means from average values for each patient. Throughout this article, n refers to the number of patients, not cells, unless otherwise stated.

Univariate ANOVA was applied to determine the sources of variation for I_K1 and I_K,ACh. Independent variables were Gß₃ genotype, selected clinical variables (Table 2), and medication (Table 3). To test for interactions between Gß₃ genotype and other parameters, interaction terms were included in separate 2-way ANOVA.

View this table: [in this window] [in a new window]   Table 2. Effects of Clinical Variables on Density of Inward Rectifier K+ Currents by Univariate ANOVA

View this table: [in this window] [in a new window]   Table 3. Effects of Medication on Density of Inward Rectifier K+ Currents by Univariate ANOVA

Differences between continuous data from patients classified by Gß₃ genotype were compared by 1-way ANOVA and post hoc multiple-comparison tests (Duncan t test) if the Lewene test for equality of homogeneity was rejected. Frequency data were analyzed with the use of likelihood ² statistics with SPSS for Windows (version 8.0). A value of P<0.05 was considered statistically significant.

	Results

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Genotyping of Patients
Among the patients in the study, the frequencies of the C and T alleles were 65% and 35%, respectively. The genotype at the 825 position was CC in 30 patients (43%), CT in 31 (44%), and TT in 9 (13%) (Table 4). Genotype distribution did not deviate from that predicted by the Hardy-Weinberg equation.¹⁴

View this table: [in this window] [in a new window]   Table 4. Patient Characteristics in Genotype Groups

With the exception of myocardial infarction status, there were no statistically significant differences with respect to sex, age, body mass index (BMI), casual blood pressure, heart rate, prevalence of ischemic heart disease, heart failure, diabetes mellitus, hyperlipidemia, or smoking across the different genotype groups (Table 4).

Associations Between Selected Clinical Variables, Medications, and Human Atrial Potassium Currents
The density of I_K1, I_K,ACh, or whole current (I_K1+I_K,ACh) was related to the 825T allele and selected clinical variables. The density of I_K1 was significantly associated only with Gß₃ genotype, whereas I_K,ACh density was also associated with BMI and smoking in addition to Gß₃ genotype (Table 2). Test of interaction effects between Gß₃ genotype and BMI or smoking on the density of I_K,ACh showed that these are independently associated with I_K,ACh, because no significant interaction was detected (P<0.155 for BMI and P<0.807 for smoking). Conversely, whole-current density was not associated with the Gß₃ genotype but rather with BMI (Table 2).

We also analyzed the effect of medication on the density of I_K1, I_K,ACh, or whole current. Long-term therapy with Ca²⁺ antagonists was associated with both the density of I_K1 and of whole current, whereas medication had no effect on density of I_K,ACh. Density of I_K1 was also related to digitalis therapy (Table 3).

Association Between Gß₃ Genotype and Human Atrial Potassium Currents
Cell size as measured by C_M was not different in the 3 genotypes: C_M was 99±6 pF for CC genotype (n=93 cells from 30 hearts), 102±4 pF for CT (n=121 cells from 31 hearts), and 100±7 pF for TT (n=32 cells from 9 hearts). Mean current amplitudes were corrected for cell size by dividing by C_M.

In a myocyte from a patient with CC genotype (Figure 1, left), I_K1 was small, resulting in a shallow curve with little indication of inward rectification. Addition of carbachol (2 µmol/L) increased inward current more than outward current; thus, inward rectification became more prominent. This behavior, ie, the relation between current amplitudes of I_K1 below and I_K,ACh above the calculated medians of all myocytes, was observed in 31.3% of cells (25/80) from CC genotype, in 26.5% of cells (31/117) from CT, and in 11.1% of cells (3/27) from TT (P=0.194, ² test). Figure 1 (right) shows a cell with a large I_K1 that exhibits a small response to carbachol. This opposite behavior, ie, the relation between current amplitudes of I_K1 above and I_K,ACh below the calculated medians, was found in TT genotype in 51.9% (14/27), in CC genotype in 23.8% (19/80), and in CT genotype in 22.2% (26/117) of myocytes (P=0.028, ² test).

View larger version (14K): [in this window] [in a new window]   Figure 1. Original tracings of IK1 and IK,ACh. Left, Control (IK1) current and current in response to 2 µmol/L carbachol (difference current taken as IK,ACh) in myocyte (CM=72 pF) from patient with CC genotype. Right, Similar tracings in myocyte (CM=113 pF) from patient with TT genotype. Inset, Clamp protocol (0.5 Hz). Only data during ramp pulse from -100 to -10 mV within 800 ms are depicted.

Cumulative concentration-response curves for carbachol were obtained in 63 myocytes from 18 patients, and individual curve fitting resulted in log EC₅₀ values of -6.76±0.17 for CC genotype (n=8 patients, 25 myocytes) and of -6.90±0.13 for CT (n=8 patients, 33 myocytes). Data from only 2 patients (5 myocytes) with TT genotype were available; the respective log EC₅₀ values were -6.85 and -6.80. These differences in log EC₅₀ values were not statistically significant.

To detect a putative association between the 825T allele and current amplitude, mean values for I_K1 and I_K,ACh were calculated for the 3 genotypes (Figure 2, left). In the inward branch (-100 mV), mean current densities (±SEM, n=patients/cells) for I_K1 were significantly larger in myocytes from TT genotype (7.46±1.14 pA/pF, n=9/32; P<0.05) than in myocytes from CC (4.98±0.49 pA/pF, n=30/93) or TC (4.25±0.36 pA/pF, n=31/121) genotype. The opposite was observed for I_K,ACh: the mean amplitudes were smaller for myocytes from patients with TT genotype (4.30±1.33 pA/pF, n=9/27; P<0.05) than those from CC (6.56±0.54 pA/pF, n=30/80) or CT (6.16±0.45 pA/pF, n=31/117) genotype. Essentially similar results were obtained with adenosine (1 mmol/L, Figure 2, right), although differences between genotypes failed to reach the level of statistical significance. In the outward current range (-10 mV), I_K1 and I_K,ACh did not vary between the 3 genotypes (data not shown).

View larger version (31K): [in this window] [in a new window]   Figure 2. Left, Mean density of IK1 and IK,ACh stimulated with carbachol (2 µmol/L) in myocytes from patients with 3 genotypes. Numbers within columns indicate number of myocytes investigated. Right, Mean density of IK1 and IK,ACh stimulated with adenosine (1 mmol/L) in myocytes from patients with 3 genotypes. IK,ACh is difference between maximum current in response to carbachol or adenosine and IK1. *P<0.05 vs IK1 in myocytes from patients with CC and TC genotype; #P<0.05 vs IK,ACh in myocytes from patients with CC and TC genotype (1-way ANOVA and Duncan’s t test).

Some myocytes were exposed to atropine (1 µmol/L) to exclude spontaneously active M₂ receptors. I_K1 was not affected by this nonselective muscarinic receptor antagonist (for 3 myocytes from 2 patients with TT genotype, 7.48±2.61 pA/pF before and 7.06±2.51 pA/pF after atropine). Similarly, glibenclamide (10 µmol/L) had no effect on I_K1 (for 3 myocytes from 1 patient with TT genotype, 9.20±2.35 pA/pF before and 7.86±2.33 pA/pF after glibenclamide), excluding a significant contribution of ATP-sensitive K⁺ currents (I_K,ATP).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The main finding of our investigation was a significant association between the 825T allele and activity of inward rectifier K⁺ currents in human atrial myocytes. The amplitude of the background inward rectifier I_K1 was enhanced in myocytes from patients with TT genotype compared with those with CC and TC genotype. In addition, we found that the increase in inward rectifier current produced by muscarinic stimulation with carbachol was reduced in homozygous 825T-allele carriers.

The original finding that the Gß₃ subunit 825T allele associates with essential hypertension³ has been confirmed in several different populations,^{4 5 6} whereas an association between Gß₃ variants and increased risk for coronary heart disease or myocardial infarction is still controversial.^{15 16} In our patients, we found no differences in clinical parameters, with the exception of myocardial infarct status, between the 3 genotypes (Table 4). When we associated the clinical parameters with current density of I_K1 and I_K,ACh, we found that I_K,ACh associates not only with C825T polymorphism but also with BMI and smoking (Table 2). However, no significant interaction effect of Gß₃ genotype and BMI or smoking on I_K,ACh was found, suggesting that these may modulate I_K,ACh independently of Gß₃ genotype. The density of whole current was not associated with Gß₃ genotype but was with BMI (Table 2). Furthermore, both I_K,ACh and whole current were associated with medication with Ca²⁺ antagonists, and I_K,ACh was associated with digitalis therapy (Table 3). However, it must be stressed that the observed associations between current density and BMI, smoking, Ca²⁺ antagonist medication, or digitalis therapy should be interpreted with great caution, because the present study was not designed to detect any of these unexpected associations, all of which should be examined separately in future studies.

I_K1 and I_K,ACh were studied with conventional voltage-clamp methods. Differences in current densities (see below) were not due to myocyte hypertrophy, because there was no difference in cell size between genotypes. Because I_K1 determines the resting membrane potential, increased density of I_K1 in right atrial myocytes from patients with TT genotype could result in more negative membrane potentials and earlier repolarization. Conversely, I_K,ACh, which is another significant contributor to resting membrane potential, was reduced in myocytes from patients with TT genotype. However, we could not actually measure differences in resting membrane potential of myocytes from different genotypes, although because of small cell numbers, this needs to be readdressed systematically.

The current density of atrial I_K1 was found to be increased in patients with chronic atrial fibrillation,¹⁷ although these changes were not detected in another study.¹⁸ Nevertheless, patients with atrial fibrillation were excluded from our study. Furthermore, atrial I_K1 is functionally downregulated in patients with heart failure¹⁹ ; however, the 3 genotypes did not vary with respect to function of the left ventricle (Table 4).

Although evidence for enhanced signal transduction in immortalized cells derived from human subjects that were 825T-allele carriers has been published previously,³ this is the first report of an attempt to associate G_i protein–mediated signaling and Gß₃ genotype in freshly isolated native cells. Considering I_K,ACh to be a sensitive indicator for the activity of G_i protein–mediated signal transduction, we expected an increased I_K,ACh. In contrast, I_K,ACh was reduced, whereas I_K1 was increased, although total current remained constant. At first sight, this finding appeared to disprove our original hypothesis, but considering the similarity of the electrophysiological properties of I_K1 and I_K,ACh on the whole-cell level, the increased background current could actually contain both current components. A contribution of I_K,ATP is unlikely, because block of these channels with glibenclamide did not affect I_K1 in myocytes from TT-genotype carriers. Thus, we suggest that the assumed I_K1 in the absence of carbachol actually may consist of unchanged I_K1 and an additional fraction of I_K,ACh stimulated by the constitutively active Gß₃ splice variant. Provided that the channel densities of I_K1 and I_K,ACh are not different between the genotype groups, this hypothesis would also explain the smaller response of I_K,ACh to carbachol, because only the channels not yet activated can still respond to receptor stimulation. Lack of effect of atropine on I_K1 excludes spontaneously active muscarinic receptors as a cause for increased background I_K1. Heterotrimers containing the short Gß₃ (Gß₃-s) subunit can be activated more effectively by G protein–coupled receptors,³ because dimers of Gß₃-s are not as tightly associated with G as unmodified ß dimers.²⁰ At the cellular level, this might create a larger pool of free ß dimers, which could activate I_K,ACh and thus explain the increase of basal activity we observed. Conversely, agonist stimulation would tremendously increase this pool of ß dimers in CC, TC, and TT genotypes. Thus, in the presence of maximally effective concentrations of receptor agonist, the channels may be saturated with ß dimers. Therefore, the response maximum could be limited by channel density, explaining the similar levels of maximum currents in cells of the different genotype groups. Alternatively, the decreased carbachol effect could be due to impaired interaction of the Gß₃-s with the corresponding -subunit. At least for M₂ receptor–mediated inhibition of Ca²⁺ channels, the subunits involved have been identified as ß₃ and ₄,²¹ and ß₃-subunits were shown to lack proper interaction with ₄-subunits in Sf9 insect cells.²² Provided that these findings can be extrapolated to atrial myocytes, impaired ß interaction could also explain the reduced response of I_K,ACh to carbachol. Further voltage-clamp experiments at the single-channel level, studies of the channel protein expression, and identification of the ß isoforms involved are necessary to distinguish between these possibilities.

Study Limitations and Clinical Implications
As in other studies in humans, the present results are limited by the small number of hearts evaluated and by the lack of control tissue. Although inward rectifier K⁺ currents are much smaller at -10 mV than -100 mV, their physiological role for repolarization is clearly larger in the outward direction. Nevertheless, we demonstrated a significant association between the Gß₃ 825T allele and either increased I_K1 or reduced I_K,ACh in myocytes from TT-genotype carriers only for inward but not for outward current direction. The increased background current density in TT carriers could shorten action potential duration. Although changes in protein expression of these channels cannot be excluded, we speculate that the increased background current density in myocytes from carriers with TT genotype in combination with reduced stimulation by carbachol could be a result of I_K,ACh being constitutively active in this genotype. Further analysis of the observed current alterations will require extensive biochemical analysis of G protein activation in human atrial myocytes. This is limited by the small amount of human tissue available from patients with TT genotype and the short viability of the cells.

	Acknowledgments

 
The authors thank Margarete Sieß, Manja Schöne, and Traulinde Großmann for their excellent technical assistance and Dr Thomas Wieland for his thoughtful comments.

Received December 31, 1999; revision received March 14, 2000; accepted March 16, 2000.

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