Mechanisms of Increased Susceptibility to Angiotensin II–Induced Apoptosis in Ventricular Cardiomyocytes of Spontaneously Hypertensive Rats
Abstract—Previous findings have shown that hypotensive doses of losartan prevent the excess of apoptosis present in the hypertrophied left ventricle of adult spontaneously hypertensive rats (SHR). This study was designed to determine whether angiotensin II facilitates apoptosis in cardiomyocytes of adult SHR. Primary cultures of ventricular cardiomyocytes from 30-week-old normotensive Wistar-Kyoto rats (WKY) and SHR with left ventricular hypertrophy were exposed to 10−9 mol/L angiotensin II for 24 hours. Apoptotic cells were assessed by terminal deoxynucleotidyl transferase assay and confirmed by Annexin V detection. The expression of Bax-α, Bcl-2, p53, and caspase-3 proteins was assessed by Western blot assays. The expression of BAX gene was assessed by Northern blot. Angiotensin II increased (P<0.01) cardiomyocyte apoptosis, and this effect was higher (P<0.001) in SHR cells than in WKY cells. Whereas losartan (10−7 mol/L) blocked the apoptotic effect of the octapeptide in cells from the two strains of rats, PD123319 (10−7 mol/L) inhibited angiotensin II–mediated apoptosis only in SHR cells. Angiotensin II stimulated (P<0.01) Bax-α protein, and this effect was higher (P<0.01) in SHR cells than in WKY cells. Angiotensin II did not modify Bcl-2, p53, and BAX mRNA in cells from the two strains of rats. Angiotensin II induced a similar increase (P<0.05) in the ratio caspase-3/procaspase-3 (an index of caspase-3 activation) in cardiomyocytes from the two strains of rats. The present in vitro results indicate that SHR cardiomyocytes exhibit enhanced susceptibility to angiotensin II–induced apoptosis. Ligand binding to angiotensin II type 1 and type 2 receptors leading to changes in posttranscriptional processing of Bax-α and accumulation of this proapoptotic protein may be involved in the abnormal response of SHR cardiomyocytes. These data support a role for angiotensin II in apoptosis observed in the left ventricle of these rats.
Increased apoptosis has been demonstrated recently in the hypertrophied left ventricle of young,1 adult,2 and aged3 spontaneously hypertensive rats (SHR). It has been suggested that apoptosis might be a mechanism involved in the reduction of cardiomyocyte mass that accompanies the transition from stable compensation to heart failure in hypertensive heart disease.4 Although the available evidence suggests that apoptosis can be induced in cardiac cells by pressure overload,5 it has been shown that cardiac apoptosis in adult SHR is not related to blood pressure but is related to overactivity of the local renin-angiotensin system.2 6
We have recently shown that an association exists between increased apoptosis and overexpression of the proapoptotic Bax-α protein in the hypertrophied left ventricle of adult SHR.6 Interestingly, long-term blockade of angiotensin II type 1 (AT1) receptors with hypotensive doses of losartan prevented Bax-α overexpression and normalized apoptosis in the left ventricle of SHR.7 Although arterial hypertension cannot be excluded as a causative factor in Bax-α overexpression and apoptosis in the left ventricle of adult SHR, the possibility also exists that the direct interaction of angiotensin II with myocardial cells plays a role in this process. This is further supported by the finding that angiotensin II induces apoptosis of adult rat ventricular cells in vitro.7 Furthermore, some findings in cultured adult cardiomyocytes suggest that angiotensin II may upregulate Bax-α protein in these cells.8
Therefore, the current work was designed to test the hypothesis that angiotensin II overstimulates Bax-α–mediated mechanisms of apoptosis in ventricular cardiomyocytes of adult SHR with left ventricular hypertrophy (LVH).
The investigation was performed according to the European Community guidelines for animal care and use of laboratory animals (Directive 86/609). The experiments were performed with rats provided by Harlan UK Limited (Blackthorn). Normotensive Wistar-Kyoto rats (WKY, n=20) and SHR (n=20) were observed in our colony until they were killed at 30 weeks of age. Systolic blood pressure (SBP) was recorded before the animals were killed by the standard tail-cuff method. In each group of animals, 10 rats were used to measure SBP and cardiac parameters and 10 rats were used to isolate ventricular cardiomyocytes. The animals were anesthetized with 30 mg/kg IP of sodium thiopental and were killed by decapitation, the heart was removed en bloc, and the cardiac dimensions were measured. In each animal, the cardiac index was calculated by dividing the heart weight by the body weight. As the equator of the heart was selected as representative of the entire left ventricle, ventricular wall thickness was measured across the free wall.
Primary cultures of adult rat cardiomyocytes were obtained by the method originally described by Jacobson and Piper,9 with some modifications. Once removed, the heart was attached to a perfusion apparatus via aortic arch. For blood washout, a calcium-free medium (115 mmol/L NaCl, 2.6 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 6 mmol/L NaHCO3, 11 mmol/L Glucose D(+), pH 7.3) was infused for 10 minutes and then collagenase A (Boehringer-Mannheim) was added at a final concentration of 0.4%. The perfusion was performed at 37°C and bubbling the medium with O2 95% and CO2 5%. During the perfusion period (35 minutes), the calcium concentration was progressively increased at 5-minute intervals from 0.25 to 1.0 mmol/L. After perfusion, the atria were discarded and the ventricles were cut in the final perfusion medium supplemented with 1.3% BSA. The suspension was slowly drawn with a 10-mL pipette, filtered through a 250-μm sterile filter, and spun down at 100g. The pellet was resuspended and slowly stirred during 10 minutes in 40 mL of purification medium (115 mmol/L NaCl, 2.6 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 6 mmol/L NaHCO3, 11 mmol/L Glucose D(+), 1 mmol/L CaCl2, 0.4% BSA, 0.025% Tripsin, pH 7.3) and centrifuged at 50g for 5 minutes. To enrich the cell suspension on rod-shaped myocytes, the pellet was resuspended on a 15-mL aliquot of enrichment medium (115 mmol/L NaCl, 2.6 mmol/L KCl, 1.2 mmol/L MgSO4, 1.2 mmol/L KH2PO4, 6 mmol/L NaHCO3, 11 mmol/L Glucose D(+), 1 mmol/L CaCl2, 4% BSA, pH 7.3) and centrifuged 3 times, discarding the supernatant. Each centrifugation was performed at 50g during 1, 2, and 3 minutes, respectively. Cells recovered from the pellet consisted of 85% to 90% rod-shaped viable cardiomyocytes. The average number of cardiomyocytes obtained from the ventricles was ≈2×106 and the percentage of non–myocyte-contaminating cells was <2%. No differences in the yield and purity of cell culture were found between WKY rats and SHR. Figure 1⇓ illustrates a representative photograph of rod-shaped cardiomyocytes in culture.
Cardiomyocytes were plated in laminin-precoated (0.5 μg/cm2) culture plates at a density of 1.5×104 cells/cm2 and incubated at 37°C in a 5% CO2 humidified atmosphere for 4 hours in cardiomyocyte-cultured medium that consisted of Medium 199 with Hanks’ salts supplemented with 26 mmol/L NaHCO3, 10−4 mmol/L insulin, 5 mmol/L creatinine, 2 mmol/L l-carnitine, 0.2% BSA, 10 μmol/L Ara-C, 5 mmol/L taurine, 100 IU/mL penicillin, 0.1 mg/mL streptomycin, 10 mmol/L N-(2-hydroxyetil)piperazine-N′2-etanolsulfonic acid) [HEPES], pH 7.4. Nonattached cells were discarded after this period and fresh culture medium was added. After 24 hours of incubation, angiotensin II was added, diluted in the culture medium at 10−11, 10−9, and 10−7 mol/L for another 24 hours. After angiotensin II treatment, cells were processed for in situ detection of apoptosis or protein extraction. In some wells assigned for apoptosis quantification, losartan or PD123319 was diluted at 10−7 mol/L as previously described10 11 in the medium, and after 1 hour of incubation, angiotensin II was incorporated.
In Situ Detection of Apoptosis
Cells for in situ detection of apoptosis were fixed by incubating with 4% formalin on ice for 20 minutes; after 3 washes with precooled Hanks’ medium, the plates were stored with absolute ethanol at −20°C until terminal deoxynucleotidyl transferase (TdT) reaction. The TUNEL methodology used for in situ end-labeling of DNA fragments was the same as recently described,6 with some modifications. Ethanol was discarded, and cells were washed 3 times with PBS (100 mmol/L NaCl, 80 mmol/L Na2HPO4, 25 mmol/L NaH2PO4, pH 7.5). TdT reaction was performed by incubating cell cultures with reaction buffer (140 mmol/L of sodium cacodylate, 30 mmol/L Tris, 1.5 mmol/L cobalt chloride [Sigma], 0.25 nmol/L deoxythymidine triphosphate, 0.25 nmol/L biotin-16-deoxyuridine triphosphate and 0.25 U/μL of TdT [Boehringer Mannheim]) at 37°C for 1 hour in a humidified chamber, and the reaction was stopped, incubating the cells with stop buffer (150 mmol/L NaCl, 15 mmol/L sodium citrate, pH 7.0) for 5 minutes. To stain the incorporated biotined nucleotides, the cells were incubated for 30 minutes at room temperature in a humidified chamber with 4 μg/mL of fluorescein-isothiocyanate (FITC)-conjugated extravidin (Sigma) diluted in the stop buffer with 0.1% Triton X-100.
A fluorescent Annexin V assay was performed to confirm the apoptotic events detected with TUNEL methodology. After washing 5 times with PBS, fixed cell cultures were incubated at room temperature for 30 minutes with Annexin V-biotin conjugated (Molecular Probes) diluted 1:80 in the Annexin V–binding solution that contained: 20 mmol/L NaCl, 2.5 mmol/L CaCl2, 10 mmol/L HEPES, pH 7.4. Annexin V–positive cells were stained with tetramethylrhodamine (TRITC)-conjugated extravidin diluted in PBS. For negative controls, deionized water was added instead of Annexin V.
After washing, cells were mounted with Vectashield mounting medium containing 1.5 μg/mL of 4′,6-diamino-2-phenylindole (DAPI) (Vector) to stain total nuclei. The analysis was performed with an epifluorescence inverted microscope equipped with 3 filters that allowed detection of DAPI-stained (excitation UV and emission blue), FITC-stained (excitation blue, emission green), and TRITC-stained (excitation green, emission red) cells. Figure 2⇓ illustrates a representative digitized image of cultured cardiomyocytes stained with the 3 fluorescent substances: DAPI, FITC, and TRITC. Cardiomyocytes exhibiting apoptotic nuclei were also positive for Annexin V staining. However, some round-shaped cells exhibiting positive staining for Annexin V and negative for TUNEL were not recorded as apoptotic cells, but they were considered to be necrotic or with membrane alterations caused by the fixation process. Similarly, few cardiomyocytes exhibiting positive staining for TUNEL and negative for Annexin V were not recorded. For quantification, a minimum of 2000 nuclei per plate were counted, and the presence of apoptotic nuclei was expressed by means of apoptotic index that was the number of apoptotic nuclei per 103 of total nuclei.
Western Blot Analysis
For immunoblot assay of Bax-α, Bcl-2, and p53 proteins in cultured cardiomyocytes, we used the same procedure recently described for the left ventricle with small modifications.6 10 Cells were harvested, washed twice in PBS, and incubated in the protein extraction buffer (1% sodium dodecyl sulfate [SDS], 0.1% Triton X-100, 5 mmol/L EDTA, 150 mmol/L NaCl, 1 mmol/L fenil metil sulfonyl fluoride [PMSF], 1 tablet of protease inhibitors cocktail [Boehringer Mannheim] per 50 mL of buffer, and 50 mmol/L Tris, pH 7.4) for 30 minutes on ice. After centrifugation for 15 minutes at 13 000 rpm in a microfuge, the supernatant was recovered and protein concentration was assessed by Bradford method. Aliquots of 50 μg were resuspended in the same volume of loading buffer (20% α-mercaptoethanol, 4% SDS, 20% glycerol, 0.0125% bromophenol blue and 0.125 mmol/L Tris, pH 6.4), boiled for 5 minutes, and size-fractionated on 12% polyacrylamide gels by electrophoresis. Bax-α, Bcl-2, and p53 immunoblot conditions were identical to those previously reported.6 12 For immunodetection of caspase-3, membranes were incubated in blocking solution (10% nonfat dry milk. 0.05% Tween in PBS) overnight at room temperature. A commercial antibody (Santa Cruz Biotechnology) that recognizes 32-kDa inactivated procaspase-3 and 20-kDa fragment of caspase-3 was used diluted at 1:500 in PBS with 1% of dry skim milk. Bound antibody was detected by a peroxidase-conjugated secondary antibody and visualized by ECL-Plus chemiluminescence detection. The optical density values were expressed as arbitrary units (AU). The caspase-3/procaspase-3 ratio was considered as an index of caspase-3 activation.
Northern Blot Analysis
Total RNA was isolated using the Trizol reagent (Gibco BRL), and 5 μg of RNA was separated in a 1.2% denaturing formaldehyde-agarose gels. Northern blotting was performed as described previously.12 The probe, a 505-bp fragment encoding for rat BAX cDNA was labeled with ([α-32P)dCTP with the Multiprime DNA labeling kit (Amersham Ibérica). After autoradiography, the relative density of each band was determined by densitometric analysis. Hybridization of the same membrane by a probe from rat GAPDH cDNA was used for normalization.
Results are presented as mean±SEM, computed from the average measurements obtained from each group of cells. Normal distribution of data were checked by means of the Shapiro-Wilks test. A Levene statistic test was performed to check the homogeneity of variances. The unpaired Student’s t test or the Mann-Whitney U test was used to assess statistical differences between cells from the two strains of rats. Differences between cells from the same strain were tested by a paired Student’s t test or by a Wilcoxon test when the Levene test was significant. Differences among cells from the same strain of rats under more than 2 experimental conditions were tested by a 1-way ANOVA. Subsequent analysis for significant differences between 2 groups was performed by means of the multiple-comparison Scheffé’s test. Probability values <0.05 were considered significant.
Blood Pressure and Cardiac Hypertrophy
As shown in Table 1⇓, SHR exhibited increased (P<0.001) values of SBP compared with WKY. Cardiac index and left ventricular wall thickness were higher (P<0.01) in SHR than in WKY (Table 1⇓). Therefore, LVH was present in SHR.
Effects of Angiotensin II on Cardiomyocyte Apoptosis
Figure 3⇓ shows the apoptotic index measured in either WKY or SHR cardiomyocytes in different experimental conditions. In basal conditions, SHR cells exhibited higher apoptotic index than WKY cells (4.85±0.81% versus 2.98±0.25%, P<0.05). Incubation with 10−9 mol/L angiotensin II increased the apoptotic index in cells from the two strains of rats (WKY cells, 5.19±0.37%, P<0.01; SHR cells, 27.82±2.7%, P<0.001). The angiotensin II–induced increase in apoptosis was higher (P<0.01) in SHR cells than in WKY cells. Exposure of cardiomyocytes to angiotensin II at concentrations >10−9 mol/L did not increase significantly the number of apoptotic cells in the two strain of rats, indicating that angiotensin II–induced cardiomyocyte apoptosis is not a concentration-dependent process. Similar results were described in cardiomyocytes10 and nonmyocytes.13 14
Preincubation with losartan blocked significantly (P<0.01) angiotensin II–induced apoptosis in cardiomyocytes from the two strains of rats. Preincubation with the AT2 antagonist PD123319 abolished (P<0.01) angiotensin II–promoted apoptosis in SHR cells but not in WKY cells. No significant differences were found between the effects of losartan and PD123319 on angiotensin II–induced apoptosis in SHR cardiomyocytes.
Effects of Angiotensin II on Bax-α and Bcl-2 Proteins
As shown in Figure 4⇓, basal expression of Bax-α was similar in WKY cells (0.85±0.07 AU) compared with SHR cells (0.85±0.07 AU). Angiotensin II induced a significant increase in Bax-α protein expression in WKY cells (1.55±0.12 AU, P<0.01) and SHR cells (2.00±0.13 AU, P<0.01) (Figure 4⇓). The magnitude of the increase in Bax-α protein induced by the octapeptide was higher (P<0.01) in SHR cells than in WKY cells. Losartan blocked (P<0.01) angiotensin II–induced Bax-α protein expression in WKY cells (1.27±0.09 AU) and SHR cells (1.16±0.09 AU) (Figure 4⇓). In addition, angiotensin II–induced Bax-α protein expression was blocked (P<0.01) by PD123319 in SHR cardiomyocytes (1.19±0.07 AU).
Angiotensin II did not modify the expression of Bcl-2 protein either in WKY cells or in SHR cells (Table 2⇓). As a consequence, the Bax-α/Bcl-2 ratio (an index of cell susceptibility to apoptosis)15 increased significantly (P<0.01) in cells from the two strains after incubation with angiotensin II (WKY cells, +51%; SHR cells, +182%). In addition, angiotensin II–induced increase in the Bax-α/Bcl-2 ratio was higher (P<0.01) in SHR cells than in WKY cells.
Effects of Angiotensin II on BAX mRNA and p53 Protein
In basal conditions, the expression of BAX mRNA and p53 protein was similar in cells from the two strains of rats (Table 2⇑). Angiotensin II did not modify the expression of these two parameters in either WKY cells or SHR cells (Table 2⇑).
Effects of Angiotensin II on Caspase-3 Activation
Figure 5A⇓ shows a representative Western blot autoradiogram of the 32-kDa procaspase-3 protein and the 20-kDa caspase-3 protein. The ratio between the optical densities measured in 20-kDa caspase-3 and 32-kDa procaspase-3 bands was calculated in each experiment as an index of caspase-3 activation.16 In basal conditions, the ratio of caspase-3/procaspase-3 was similar in WKY cardiomyocytes (0.13±0.01) and SHR cardiomyocytes (0.14±0.01). Angiotensin II increased (P<0.05) the caspase-3/procaspase-3 ratio in both WKY cells (0.21±0.03) and SHR cells (0.24±0.02), the magnitude of the increase being similar in the two strains of rats.
The main finding of this study is that angiotensin II induces an exaggerated apoptotic response in cardiomyocytes from the hypertrophied left ventricle of adult SHR compared with ventricular cardiomyocytes from adult WKY. Furthermore, whereas angiotensin II–induced apoptosis was prevented by AT1 blockade in WKY cells, blockade of both the AT1 and the AT2 receptor blunted the apoptotic response to the octapeptide in SHR cardiomyocytes.
Hamet et al17 have shown that isoproterenol significantly increased apoptosis in cultured aortic smooth muscle cells from normotensive Brown Norway rats and SHR, with higher increments in SHR versus Brown Norway vascular smooth muscle cells. Thus, our finding in cardiomyocytes would support the notion that in cardiovascular cells of genetically hypertensive rats exists an increased susceptibility to apoptotic stimuli (ie, vasoactive agonists).
Although AT1 receptor antagonism has been shown to block angiotensin II–induced apoptosis in cultured ventricular cardiomyocytes from neonatal10 18 and adult7 normotensive rats, AT2 receptor antagonism did not exert any influence. In addition, it has been reported that adult rat cardiomyocytes submitted to maneuvers that activate cellular angiotensin II exhibited increased apoptosis inhibitable by pretreatment of cells with an AT1 antagonist.8 19 20 It thus appears that angiotensin II–induced apoptosis in cardiomyocytes from adult nonhypertensive rats is mediated by the AT1 receptor–dependent pathway.
The variation in the relative ratio of AT1 receptor expression to AT2 receptor is regulated to different extents in physiological and pathological conditions. Fetal and neonatal cardiomyocytes have both types of receptors, but the proportion of AT1 on the cells progressively increases with maturation, whereas the AT2 receptor almost disappears in adult ventricular cardiomyocytes.21 In several experimental models of cardiac hypertrophy associated with hemodynamic overload including SHR with LVH,22 substantial increases in ventricular AT2 receptors with a decreased AT1-to-AT2 relative ratio have been described. Therefore, although we have not measured the density of angiotensin II receptors, it is conceivable that SHR cells but not WKY cells express AT2 receptors. Because angiotensin II has been shown to induce apoptosis in other cardiovascular cell types through stimulation of AT2 receptors,23 it can be proposed that the participation of both AT1- and AT2-dependent apoptotic pathways may account for the exaggerated apoptotic response to the octapeptide in SHR cardiomyocytes. Furthermore, some kind of cross-talk between the two receptors may exist that mediates apoptosis in SHR cardiomyocytes. This is supported by our finding that whereas angiotensin II stimulates 5.7-fold apoptosis when the two receptors are exposed, the ability of the octapeptide to stimulate cardiomyocyte apoptosis is strongly reduced when one of the two receptors is blocked.
A second finding of the current study is that angiotensin II induces the expression of the proapoptotic protein Bax-α in cardiomyocytes, this effect being much more marked in SHR cells than in WKY cells. Again, this effect was blocked by losartan in WKY cells and by losartan and PD123319 in SHR cells. Thus, overstimulation of a Bax-α–mediated apoptotic pathway may be involved in apoptotic hyperresponsiveness of SHR cardiomyocytes to angiotensin II. This is further supported by our finding that the increment in the Bax-α/Bcl-2 ratio (an index of cell susceptibility to apoptosis)15 induced by angiotensin II is much more higher in SHR cells than in WKY cells.
A number of experiments have shown that maneuvers that stimulate intracellular angiotensin II also induce Bax-α protein in cardiomyocytes. For instance, p53-transfected cardiomyocytes exhibit increased apoptosis and overexpress Bax-α protein in association with the activation of cellular renin-angiotensin system.20 Furthermore, stretch-mediated release of angiotensin II in vitro promotes cardiomyocyte programmed cell death, and this phenomenon is associated with increase in Bax-α protein.8 The same authors have shown that cardiomyocyte stretching was coupled with enhanced p53-dependent BAX mRNA and protein expression,8 thus suggesting that stimulation of BAX gene transcription may account for enhanced Bax-α protein. Although we have not measured p53 binding activity, our results of normal levels of p53 protein and BAX mRNA do not support a role for the above mechanism of enhanced Bax-α protein in cardiomyocytes exposed to extracellular angiotensin II. Thus, abnormalities in the posttranscriptional regulation of Bax-α (ie, activation of protein kinases, inhibition of phosphatases, decrease in intracellular pH)24 leading to diminished proteolytic degradation may account for overexpression of the protein observed in SHR cardiomyocytes exposed to the octapeptide.
Another finding of this study is that angiotensin II increases the caspase-3/procaspase-3 ratio in ventricular cardiomyocytes from the two strains of rats. Because caspase-3 exists as zymogen that must first be proteolytically cleaved to become activated protease,25 this ratio has been used as an index of caspase-3 activation.16 We thus demonstrate for the first time that angiotensin II activates caspase-3 in adult rat cardiomyocytes. On the other hand, it has been shown that overexpression of Bax protein in mammalian cells is accompanied by release of mitochondrial cytochrome C, dimerization of apoptotic protease activating factors (APAF) 1, activation of caspase-3, cleavage of the DNA repair enzyme poly-ADP-ribose-polymerase (PARP), DNA fragmentation, and apoptosis.26 Therefore, it can be proposed that angiotensin II–induced overexpression of Bax-α protein may activate a caspase-3–dependent effector mechanism of apoptosis in cardiomyocytes.
Interestingly, despite a similar activation of caspase-3, the final apoptotic response induced by angiotensin II was greater in SHR cardiomyocytes than in WKY cardiomyocytes. This suggests that caspase-3–independent effector mechanisms of apoptosis can be operating in SHR cardiomyocytes. In support of this possibility are experiments showing that overexpression of Bax protein in stable transfected Jurkat cells results in alterations in mitochondrial function and subsequent apoptosis that do not apparently require caspase-3 activation.27 28
In conclusion, the results of this study indicate that the apoptotic response to angiotensin II is higher in SHR cardiomyocytes than in WKY cardiomyocytes. Ligand binding to the AT1 and the AT2 receptor and the subsequent accumulation of the proapoptotic protein Bax-α may account for the abnormal response of SHR cardiomyocytes to the octapeptide. These in vitro data support the hypothesis that increased susceptibility of SHR cardiomyocytes to the apoptotic action of angiotensin II may contribute to enhanced apoptosis seen in vivo in the hypertrophied left ventricle of these rats. Furthermore, our data add further support to the hypothesis by Hunter and Chien29 that biomechanical stress, such as chronic hypertension and mechanical load, activates multiple parallel and converging signals for cardiac hypertrophy and apoptosis, which represent two distinct outcomes in individual cardiomyocytes.
- Received April 5, 2000.
- Revision received May 3, 2000.
- Accepted June 12, 2000.
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