Increased contractility and altered Ca2+ transients of mouse heart myocytes conditionally expressing PKCbeta

Lin Huang1, Beata M. Wolska1,2, David E. Montgomery1, Eileen M. Burkart1, Peter M. Buttrick2, and R. John Solaro1

1 Department of Physiology and Biophysics and 2 Section of Cardiology, Department of Medicine, Program in Cardiovascular Sciences, College of Medicine, University of Illinois at Chicago, Chicago, Illinois 60612


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Activation of protein kinase C (PKC) in heart muscle signals hypertrophy and may also directly affect contractile function. We tested this idea using a transgenic (TG) mouse model in which conditionally expressed PKCbeta was turned on at 10 wk of age and remained on for either 6 or 10 mo. Compared with controls, TG cardiac myocytes demonstrated an increase in the peak amplitude of the Ca2+ transient, an increase in the extent and rate of shortening, and an increase in the rate of relengthening at both 6 and 10 mo of age. Phospholamban phosphorylation and Ca2+-uptake rates of sarcoplasmic reticulum vesicles were the same in TG and control heart preparations. At 10 mo, TG skinned fiber bundles demonstrated the same sensitivity to Ca2+ as controls, but maximum tension was depressed and there was increased myofilament protein phosphorylation. Our results differ from studies in which PKCbeta was constitutively overexpressed in the heart and in studies that reported a depression of myocyte contraction with no change in the Ca2+ transient.

hypertrophy; signal transduction; myofilaments; conditional transgenic; protein kinase C


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

IN EXPERIMENTS PRESENTED HERE, we used a binary transgenic (TG) mouse model in which a constitutively active beta -isoform of protein kinase C (PKC) was conditionally expressed at a low level in the heart. PKCbeta is normally expressed in fetal heart, declines after birth (20, 21), and remains low in abundance throughout normal adult life (2). There are, however, several lines of evidence indicating that the PKCbeta isoform is upregulated during development of cardiac hypertrophy. Hypertrophy induced by pressure overload in rats is associated with an increased level of PKCbeta isoform expression (12). Moreover, samples of failed human heart demonstrate an increase in PKCbeta expression and an increase in its contribution to total PKC activity (3).

To test the hypothesis that upregulation is an important element in the hypertrophic pathway, increased levels of the PKCbeta isoform have been induced using TG approaches. Wakasaki et al. (27) reported that cardiac- specific and robust overexpression of the PKCbeta isoform in TG mice induced a cardiomyopathy, which was characterized by left ventricular hypertrophy, myocardial fibrosis, and decreased in vivo left ventricular performance. However, Bowman et al. (4), who employed conditional and relatively low expression of the PKCbeta transgene in adult mice, reported that modest upregulation caused mild and progressive ventricular hypertrophy without significant pathological changes. Compared with controls, these hearts did, however, exhibit reduced rates of rise and fall of left ventricular pressure in open-chest preparations. In contrast, overexpression of PKCbeta in newborns resulted in sudden death marked by abnormalities in the regulation of intracellular Ca2+ in myocytes. In the case of mouse hearts subjected to high levels of constitutive expression of the PKCbeta isoform, Takeishi et al. (26) reported that myocyte shortening was depressed with no change in the peak amplitude of the Ca2+ transients. Associated with this effect was an increase in troponin I (TnI) phosphorylation. This finding led to the speculation that a reduction in myofilament activation, previously shown to be caused by PKC-dependent phosphorylation of cardiac troponin I (cTnI) (14, 18, 19), may be a critical mechanism for heart failure. Thus the mechanism by which PKCbeta may be involved in the hypertrophic-failure process appears to depend on the timing of the upregulation of the enzyme as well as on variable effects of upregulation on Ca2+ homeostasis and on myofilament response to Ca2+.

To test this idea, we induced expression of PKCbeta in TG mice at 10 wk of age and studied ventricular preparations at 6 and 10 mo of age. We compared preparations from controls and TG hearts with regard to 1) mechanical function and intracellular Ca2+ transients of ventricular myocytes, 2) protein phosphorylation profiles of cellular proteins, 3) Ca2+ dependence of tension in skinned fiber bundles, and 4) Ca2+ transport rate of sarcoplasmic reticulum (SR) vesicles. Our results indicate that low-level expression of PKCbeta , which may mimic a potential early response to upregulation in hypertrophy, induced changes in cellular Ca2+ regulation that are quite different from those seen in later, more malignant, stages of the pathology.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

TG animals. Conditional expression of a constitutively active PKCbeta isoform was carried out using a tetracycline-controlled promoter (4, 11). PKCbeta expression was turned on at the age of 10 wk in binary TG [tTA/PKCbeta , which we will refer to as TG (+/+)]. Controls were wild-type mice or single TG, tTA/PKCbeta (+/-), that do not express PKCbeta . As previously reported (4), preparations from single (+/-) and nontransgenic (NTG) mouse hearts differ in no significant way. The genotype of each animal was determined by either PCR or Southern blotting. Results of comparative determinations of the overall PKC activity indicate that the TG hearts have an ~10% increase in activity over the controls. Quantitative RT-PCR (using primers with matched transmembrane segments and real-time fluorescent PCR) has shown that the message level of the transgene was approximately five- to tenfold higher than the endogenous gene and that expression of the transgene does not change the level of expression of the wild-type genes.

Isolation of myocytes. Adult mice were heparinized (5,000 U/kg body wt), and, after 30 min, anesthetized with ether. Left ventricular myocytes were isolated as previously described (32) and were studied 1-6 h after isolation. The cells used for phosphorylation experiments were sequentially resuspended in 0.2, 0.5, and 1.0 mmol/l Ca2+ in a sodium HEPES phosphate-free buffer (pH 7.4) of the following composition (in mmol/l): 4.8 KCl, 1.2 MgSO4, 132 NaCl, 10 HEPES, 2.5 sodium pyruvate, and 10 glucose.

Measurement of intracellular Ca2+ transients and cell shortening. After isolation, the cells were loaded for 15 min at room temperature with 3 µmol/l fura 2-AM in Tyrode solution (0.5 mmol/l Ca2+) with 1 mg/ml bovine serum albumin and 5% fetal bovine serum. Fura 2 fluorescence and cell shortening were monitored simultaneously, as described in detail by Wolska et. al (33). Most cells were stimulated at 0.5 Hz, but in one series of experiments, we measured cell shortening over a frequency range of 0.25-3 Hz, with stimulation at each frequency for 30 s, at which time the contractions were stable.

Labeling of mouse myocytes with 32P and determination of protein phosphorylation. The level of protein phosphorylation in myocyte preparations was measured using a protocol modified after that described by Wolska et al. (33). Myocytes were incubated for 1 h in a phosphate-free sodium HEPES buffer that contained 1 mM CaCl2 and 0.250 mCi [32P]orthophosphate. Myocytes were washed twice and solubilized in 40 µl of 1% SDS containing 3 mM EDTA and sonicated for 2 min. The samples were boiled for 10 min before 12.5% SDS-PAGE. Each lane was loaded with 25 µg of protein. The gels were stained for 2 h. Destained gels were exposed to a phosphor screen overnight using STORM (Molecular Dynamics) and ImageQuant software to determine the 32P incorporation after background correction. We used a densitometric scan of the Coomassie blue-stained gel to test for equal loading of the myofilament proteins. Actin and tropomyosin were used as standards and were not statistically different in the NTG and both TG lanes. Therefore, we concluded that any changes in the phosphorylation of the myofilament proteins were the result of increased phosphorylation by PKCbeta .

Force measurements on skinned fiber bundles and Ca2+ uptake by SR vesicles. Measurements of the relationship between pCa (-log of the molar free Ca2+) and tension (force/cross-sectional area) were performed on detergent-extracted fiber bundles as described in detail previously (8, 31). The only modification was addition of the phosphatase inhibitor calyculin A (0.1 µM) to the buffers. Ca2+ uptake into SR vesicles in cardiac homogenates was measured with the aid of 45CaCl2 using a Millipore filtration assay as previously described (8, 22).

Statistical analysis. Data are presented as means ± SE. The Student's t-test was used for unpaired observations and one-way ANOVA for multiple comparison. P < 0.05 was considered significant.


    RESULTS
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INTRODUCTION
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RESULTS
DISCUSSION
REFERENCES

Cell shortening and Ca2+ transients in myocytes. Morphological and mechanical properties, as well as stability of the Ca2+ transients, were used as criteria for the viability and stability of the myocytes. Before making measurements, all myocytes were stimulated at 0.5 Hz for at least 20 min or until the shortening was stable for 15-20 min. Representative recordings of myocyte mechanics and Ca2+ transients in cells from 6-mo-old control and TG mice are shown in Fig. 1. As summarized in Table 1, the percentage of the extent of myocyte shortening (%l), as well as the rate of shortening and rate of relengthening, was significantly (P < 0.01) increased in TG (+/+) myocytes compared with controls. Table 1 also indicates that the time required for the cells to achieve 80% of full relaxation from the peak of the shortening signal was reduced significantly in the TG (+/+) mice compared with controls. Table 2 reports data indicating that the amplitude of the Ca2+ transient was significantly increased in the TG (+/+) myocytes. The baseline Ca2+ level was not different between control and TG (+/+) mice. There was also a significant reduction in the time to 50% decay of the Ca2+ signal from its peak in the TG (+/+) mice compared with controls (Table 2). These data show that myocytes isolated from hearts conditionally expressing low level PKCbeta demonstrated an increase in contractility.


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Fig. 1.   Representative recordings of mechanics and Ca2+ transients of myocytes isolated from 6-mo-old control and binary (+/+) transgenic (TG) mouse hearts expressing protein kinase C beta  (PKCbeta ). Top: Ca2+ transients. Middle: cell shortening. Bottom: rate of shortening.


                              
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Table 1.   Properties of isolated myocytes from 6-mo-old binary and control mouse hearts


                              
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Table 2.   Ca2+ transients of isolated cardiomyocytes from 6-mo-old binary and control mouse hearts

Data summarized in Table 3 compare results of measurements on myocytes isolated from TG mouse hearts at 10 mo of age with age-matched controls. These myocytes demonstrated increases in extent and velocity of shortening similar to those found at 6 mo of age. Figure 2 illustrates the dependence of these changes on frequency of stimulation over a range from 0.25 to 3 Hz. Both control and TG (+/+) cells showed a negative frequency-shortening relationship. At frequencies of 0.25, 0.5, 1.0, and 2.0 Hz, the percent shortening and the rate of relengthening were increased compared with these same parameters for control myocytes. At a stimulation frequency of 3 Hz, there was no difference in percent shortening and the rate of relengthening between the two groups (Fig. 2). Thus the effect of PKCbeta expression was sustained over a broad range of frequencies of stimulation. The heart weight-to-body weight ratio for 10-mo-old mouse TG PKCbeta hearts was 8.2 ± 0.6 mg wet heart wt/g body wt and 6.0 ± 0.2 for the controls. Thus the TG hearts demonstrated a significant (P < 0.01) hypertrophy.

                              
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Table 3.   Properties of isolated myocytes from 10-mo-old mouse heart



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Fig. 2.   Frequency-shortening relationship in control and TG (+/+) mouse myocytes. Data are presented as means ± SE (n = 8 cells). *Significantly different from controls.

Myofilament tension generation. Figure 3 shows the pCa-tension relations of skinned fiber bundles prepared from 10-mo-old binary TG (+/+) and control mouse ventricles at sarcomere length 2.3 µm. Tension generated by these two groups, which was measured in the presence of the phosphatase inhibitor calyculin A, was equally sensitive to Ca2+. The pCa50 for binary TG (+/+) preparations was 5.54 ± 0.01 (n = 13 from 4 different hearts) and 5.53 ± 0.02 (n = 13 from 4 different hearts) for controls. However, maximum tension was significantly reduced (P < 0.01) in the preparations from binary mice (34.6 ± 1.6 mN/mm2) compared with the controls (46.6 ± 2.2 mN/mm2). If both preparations were permitted to dephosphorylate, this difference in maximum tension was no longer evident (data not shown). Thus we cannot ascribe the difference in maximum tension to a difference in the myosin content of the preparations.


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Fig. 3.   pCa-tension relationship of skinned fiber bundles prepared from TG (+/+) and from control mouse hearts at sarcomere length 2.3 µm. Data are presented as means ± SE for 13 determinations from 4 control hearts and for 13 determinations from 4 TG hearts. *Significantly different from control.

Phosphorylation of cardiac proteins. To determine whether differences in covalent modulation among groups, NTG (-/-), TG binary (+/+), and single TG (+/-) could account for the decrease in maximum tension, we measured phosphorylation of the cellular proteins. Figure 4 shows an autoradiogram of the cell proteins from the three groups. There were significant increases in the 32P incorporation into myosin binding protein C (MyBPC; 25%), troponin T (TnT; 39%), TnI (20%), and myosin light chain 2 (MLC2; 60%) in the binary (+/+) TG myocyte preparations, compared with the wild-type (-/-) and the single TG (+/-), which were not different from each other. On the other hand, there was no difference in the incorporation of 32P into phospholamban.


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Fig. 4.   Autoradiogram illustrating 32P labeling of cellular proteins in myocytes from wild-type (-/-), binary TG (+/+), and single TG (+/-) mouse hearts. Each lane was loaded with 25 µg of protein. Myofilament proteins were identified by comigration with known standards or with specific antibodies. The 32P incorporation was increased in all 4 known myofilament substrates for PKC in the binary TG (+/+) preparations compared with controls. However, there was no change in 32P incorporation into phospholamban.

SR Ca2+-uptake rates. The effects of PKC expression on the initial rates of ATP-dependent SR Ca2+ uptake facilitated with oxalate were assessed using cardiac homogenates. Figure 5 shows the initial rates of Ca2+ uptake by vesicles of SR, which were assayed at various Ca2+ concentrations. The maximum uptake rate by the SR vesicles of TG mouse hearts (421.9 ± 28.1 nmol · mg-1 · min-1) was not significantly different than the uptake rate of controls (441.5 ± 27.4 nmol · mg-1 · min-1). EC50 values for Ca2+ dependence of uptake rates were 6.49 ± 0.06 µM for controls and 6.51 ± 0.05 µM for TG.


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Fig. 5.   Ca2+ uptake of sarcoplasmic reticulum (SR) vesicles in ventricular homogenates. Determination of initial rates of Ca2+ uptake by SR vesicles in mouse cardiac homogenates from transgenic (n = 4) and wild-type (n = 4) mice was made as a function of Ca2+ concentration. There were no significant differences between controls and TG (+/+) binary mice expressing PKCbeta . Data are presented as means ± SE.


    DISCUSSION
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ABSTRACT
INTRODUCTION
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Using a TG model that allows conditional expression of PKC in the heart, we have been able to shed light on the genesis of hypertrophic process and postulate how failure may follow. The major novel finding of our study is that physiologically relevant, cardiac-specific expression of PKC, initiated in adult life, produces significant inotropic enhancement, as measured by unloaded rates of cell shortening and relaxation. These changes are mirrored by increases in the dynamics and peak amplitude of the Ca2+ transient and are associated with significant cellular hypertrophy. It is also apparent that these changes occur against a background of reduced maximum tension-generating capability of the myofilaments with no change in myofilament Ca2+ sensitivity. This scenario is in sharp contrast with the changes induced by constitutive overexpression of PKCbeta in which there is a 20-fold increase in expression of cytoplasmic PKC and a 10-fold increase in bound PKC (26, 27). These models demonstrate high levels of PKC expression and activity from birth through adult life and induce depressed myocyte contractility without change in the dynamics of the Ca2+ transient. Together, these two lines of evidence indicate that there may be a hierarchy of PKC effects in the adult cardiocyte, initially characterized by a balance of increased cellular Ca2+ availability and depressed myofilament activity in association with mild, well-compensated hypertrophy. Our data also indicate that the balance of responses to PKC activation may change in favor of depressed myofilament activity with the progression from compensated hypertrophy to decompensated heart failure.

The cellular address where enhancement of Ca2+ fluxes resides appears to be the sarcolemma and not the SR, inasmuch as we were unable to demonstrate altered rates of Ca2+ uptake by vesicles of the SR in preparations from TG hearts. We cannot, however, rule out a possible effect of PKC on Ca2+ release via the ryanodine receptor (13, 16). L-type Ca2+ channels are likely candidates for the action of PKCbeta . There are several reports demonstrating that PKC phosphorylation enhances Ca2+ influx during the action potential. Moreover, PKC activators, such as phorbol esters and diacylglycerols, increase Ca2+-channel currents in cardiac and smooth muscle cells of various mammals (6, 34). In addition, our own data from studies on neonatal cardiocytes expressing the PKCbeta transgene demonstrate both a prolongation of the Ca2+ transient (4) and also an increase in the Ca2+ conductance through the L-type Ca2+ channel that is blocked by a specific PKCbeta 2 antagonist (1).

An alternate mechanism by which PKC might affect contractility is by PKC-dependent phosphorylation of the myofilament proteins cTnI, cardiac TnT (cTnT), MLC2, and MyBPC (23). Our results represent the first explicit determination of the net effect of protein phosphorylation on myofilament response to Ca2+ in a TG model of PKC expression. Our data show a depression in maximum tension with no effect on Ca2+ sensitivity. Earlier studies, including our own, have investigated effects of specific phosphorylation of the main sites: cTnI, cTnT, MyBPC, and MLC2. Among these sites, PKC-dependent phosphorylation of only two, cTnI and cTnT, have been reported to depress maximum actin-activated myosin ATPase rate and maximum cross-bridge binding to the thin filament, even at saturating Ca2+ (14, 18, 19). Also, phosphorylation of MLC2 has no effect on maximum tension (17). Phosphorylation of TnI may reduce Ca2+ sensitivity (29), and phosphorylation of MLC2 may increase Ca2+ sensitivity of force (17) and thus have offsetting effects. This finding is significant in that it demonstrates that when both are phosphorylated, covalent modulation of cTnI may outweigh the impact of modulation of MLC2 and impose a depression of myofilament of Ca2+ sensitivity. We (9, 29) could find no effect of MyBPC-protein phosphorylation on Ca2+ sensitivity or ATPase and maximum tension. On the other hand, phosphorylation of MyBPC may possibly be related, in part, to the enhanced rate of shortening of the isolated myocytes (30).

Our data indicate that at different stages of cardiac hypertrophy, there is a shifting balance between the positive effects of PKC on the Ca2+-channel conductance and the depressive effects of PKC activation on the myofilaments. Our conclusion from the studies presented here is that the increase in the amplitude of the Ca2+ transient overrides the depression in maximum tension. The depression in maximum tension was an ~25% decrease, whereas there was an 85% increase in the rate of cell shortening (+dL/dt) of the myocytes and an increase of 160% in the amplitude of the Ca2+ transient. The steepness of the relationship between Ca2+ and tension would indicate that this increase in Ca2+ delivery to the myofilaments would indeed increase contractility, despite a blunted maximum tension. It remains a distinct possibility that the increased Ca2+ could also trigger and promote the other features of the hypertrophic phenotype, including PKC activation of phosphorylation cascades affecting transcription (25). In models of heart failure in which sustained activation of PKC can be demonstrated (including prolonged insulin-deficient diabetes, end-stage cardiomyopathy, and TG constitutive expression of PKC), we propose that this balance may be tilted in favor of the negative inotropic effects of myofilament protein phosphorylation, especially of cTnI and cTnT. This proposal fits with data of de Tombe (7), who reported that the early hypertrophic response following myocardial infarction in rats was associated with a normal Ca2+-tension relationship in skinned trabeculae. However, aftersigns of congestive heart failure had become manifest; the Ca2+-tension relationship was rightshifted and depressed, compared with sham-operated controls. Moreover, previous studies (4) comparing controls with the TG model employed in the present study reported a depression in maximum pressure development in left ventricles of open-chest mice 9 mo after the PKCbeta gene was turned on. This result fits with our finding (Fig. 3) of a depression in maximum tension-generating capability of the myofilaments 10 mo after PKCbeta was turned on.

What is not directly addressed in the present study is the role played by specific isoforms of PKC. In the adult mouse heart, the prominent isoforms are Ca2+ independent (epsilon  and delta ), although Ca2+-dependent isoforms are also seen (24). Whether the beta -isoform is expressed in nonpathological adult cardiac myocytes is controversial. However, during fetal life and in response to pathological loads, increased expression is seen (10). The cellular functions subserved by individual isoforms are not established. PKCepsilon appears to translocate to the sarcomere when activated and has been shown to have a high affinity for TnI (14). In contrast, phosphorylation of the L-type Ca2+ channel has been linked to the Ca2+-dependent alpha - and beta -isoforms (35).

Despite extensive data from in vivo and in vitro experiments linking PKC activation to the development of pathological cardiac adaptations, the mechanism(s) by which kinase activation induces a hypertrophic response remains elusive. PKC influences a number of intracellular processes, including transcriptional transactivation of late response genes (25), alterations in cellular Ca2+ fluxes (5, 15, 28), and altered response of the myofilaments to Ca2+ (11, 14, 19). Because it is difficult to recapitulate the in vivo situation on isolated cells or protein preparations, defining the relevance of each of these effects has been aided greatly by TG approaches. We have investigated a novel TG mouse model to establish that low levels of PKCbeta activation in the adult cardiocyte are sufficient to cause cardiac hypertrophy. The hypertrophy is characterized by an increase in the amplitude of the Ca2+ transient and increased rates of cell shortening, despite a depression of maximum tension-generating capability by the myofilaments. In contrast, more substantial increases in transgene expression and enzyme activation have been reported to induce reduced rates of cell shortening and a rightshifted Ca2+-tension relationship associated with in vivo evidence of heart failure (26). Our findings support the hypothesis that there is a hierarchy of effects of enzyme activation in the cardiocyte and that the progression from compensated to uncompensated hypertrophy and failure may be a reflection of the intensity or duration of activation of this second messenger pathway.


    ACKNOWLEDGEMENTS

We are grateful to Ron McKinney for technical assistance in carrying out these experiments.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Research Grants R37-HL-22231 (to R. J. Solaro), R01-HL-64035 (to R. J. Solaro), R01-HL-52230 (to P. M. Buttrick), and R29-HL-58591 (to B. M. Wolska). L. Huang and E. M. Burkart were supported in part by National Institutes of Health Training Grant T32-07692. E. M. Burkart is also the recipient of an American Heart Association Midwest Affiliate predoctoral fellowship.

Address for reprint requests and other correspondence: R. J. Solaro, Dept. of Physiology and Biophysics (M/C 901), College of Medicine, Univ. of Illinois at Chicago, 835 S. Wolcott Ave., Chicago, IL 60612-7342 (E-mail: solarorj{at}uic.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 17 October 2000; accepted in final form 15 December 2000.


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RESULTS
DISCUSSION
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