The heat shock protein heme oxygenase-1 (HO-1) is regulated by a variety of physiological and pharmacological factors. In skeletal muscle tissue, HO-1 has been shown to be induced only by exercise and electrical stimulation in vivo. Both hemin and sodium nitroprusside (SNP) are potent inducers of HO-1 in other tissues. In this study, we examined the effects of these two agents on HO-1 induction in L6.G8 rat skeletal myoblast cells. Hemin and SNP increased cellular heme oxygenase activity in both a time- and concentration-dependent manner. Increases in the HO-1 mRNA level and protein expression accompanied changes in heme oxygenase activity. The ability of SNP to induce HO-1 in L6.G8 cells was reduced by coincubation with hydroxocobalamin, a known nitric oxide (NO) scavenger, suggesting that NO itself may be involved in HO-1 gene stimulation. These results indicate that HO-1 expression is sensitive to both hemin and SNP in skeletal myoblast cells and may indicate an important regulatory mechanism of heme catabolism in skeletal muscle tissue.
- heme proteins
- heat shock protein 32
the generation of free radical species during oxidative stress has been implicated in the pathogenesis of cellular damage (11). Free heme is a major source of iron that contributes to the generation of hydroxyl radicals by the Fenton reaction (7). Heme itself is released from intracellular heme proteins during increased free radical production (24). Although the precise source of this free heme is uncertain, the following sources have been suggested: denaturation of intracellular heme proteins by free radicals, newly synthesized heme not utilized for de novo heme protein assembly because redox changes in the cell prevent constitutive protein formation, and circulating hemoglobin that has entered the cell following membrane damage (14). In addition to these, certain cell types have very high levels of an alternative source of intracellular heme, namely, erythrocytes, in the form of hemoglobin, and myocytes, in the form of myoglobin. The regulation of heme turnover in these cells is therefore of particular interest.
Heme oxygenase is a microsomal enzyme, widely distributed in mammalian tissues, which has a major role in heme metabolism. It catalyzes the oxidation of heme to biliverdin and carbon monoxide (13). Two isoforms have been identified that are separate gene products: heme oxygenase-1 (HO-1), the inducible form (also known as heat shock protein 32), and HO-2, the constitutive form (16). Recently, evidence of a second constitutive isoform (HO-3) has been found (12). The role of heme oxygenase in different tissues has not, as yet, been fully characterized, but it is becoming evident that it is involved in a variety of cellular regulatory and protective mechanisms. HO-1 has been shown to be protective against ischemia-reperfusion and free radical damage in a number of tissues (15, 22, 36). Several possible mechanisms for this protection have been proposed:1) elimination of the pro-oxidant free heme could decrease hydroxyl radical formation, thereby reducing cellular damage; 2) biliverdin and the reduced product bilirubin are both potent free radical scavengers with antioxidant properties (32); and3) carbon monoxide, as a vasodilator (6), could play an important role in maintaining cellular integrity and function under certain conditions such as ischemia (14). The relative importance of each of these factors in cytoprotection under different pathophysiological conditions has not been established but is currently under investigation.
Myoglobin, a potential source of free heme, is released from skeletal muscle during severe ischemia-reperfusion injury and is implicated in the pathogenesis of acute renal failure. The precise mechanism of this is unclear; however, both iron released from heme (23) and H2O2generation mediated by heme (27) have been proposed as mediators of the damage. Induction of HO-1 in the kidney has been shown to protect against this form of renal injury (20). Whether a similar mechanism of injury occurs in the muscle itself during ischemia-reperfusion and whether HO-1 has a protective role in this tissue are not known.
The HO-1 isoform is extremely sensitive to a variety of agents that cause oxidative stress, including heat shock (29), ischemia (15), hypoxia (19), and endotoxin (36). It is also inducible by hemin (31, 33, 37) and the free radical nitric oxide (NO) (3, 5, 17). HO-1 is induced in rat skeletal muscle following exhaustive exercise and electrical stimulation (4); however, no other inducer of HO-1 in skeletal muscle has been identified. We set out to investigate the effect of two known inducers [hemin and sodium nitroprusside (SNP), an NO donor] on the expression of HO-1 in skeletal myoblast cells to establish whether its regulation is similar to that of other cell types.
MATERIALS AND METHODS
Hemin was obtained from Porphyrin Products (Logan, UT). Rabbit polyclonal antibody to HO-1 was obtained from StressGen (Victoria, BC, Canada). All other reagents were obtained from Sigma-Aldrich (Poole, UK).
Cell culture and incubations.
L6.G8 rat skeletal muscle myoblasts were obtained from the European Collection of Animal Cell Cultures (Salisbury, UK). Cells were seeded at a density of 106 cells/ml in 75-cm2 culture flasks and grown to confluence in complete medium (DMEM supplemented with 10% heat-inactivated fetal bovine serum, 2 mMl-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin). Flasks were kept at 37°C in a humidified atmosphere of air and 5% CO2. To determine the effect of hemin and SNP on the expression and activity of heme oxygenase in myoblast cells, the complete medium was replaced with incubation medium. This was made by preparing stock solutions of hemin, SNP, hydroxocobalamin (HCB), or cadmium chloride, which were then sterilized through a 0.22-μm filter and diluted to the desired concentration in complete medium. Serum-rich medium was used throughout to minimize variables and prevent HO-1 induction by stress caused by alteration of the serum content of the incubation medium (35).
Three sets of experiments were performed. The first was to determine the time dependence of heme oxygenase activity in myoblast cells. Cells were incubated for various durations (0–24 h) in incubation medium containing either 200 μM hemin or 0.5 mM SNP. The second set of experiments was to determine the concentration dependence of heme oxygenase activity and HO-1 expression in myoblast cells. Cells were incubated for 6 h in incubation medium containing various concentrations of either hemin (0–400 μM) or SNP (0–2 mM). A third set of experiments was performed to investigate the mechanism of induction of HO-1 by SNP. In addition to NO, SNP is known to release iron, cyanide, and oxygen free radicals (26), all of which may potentially induce HO-1. HCB was used as a scavenger of NO to determine the contribution of NO to SNP-mediated HO-1 induction (25). To exclude other possible antioxidant effects of HCB, cells were also exposed to another inducer of HO-1, cadmium (8). Cells were incubated for 6 h in incubation medium containing either SNP (0.5 mM) or cadmium chloride (10 μM), with or without HCB (1 mM).
Measurement of heme oxygenase activity.
Cells were manually scraped from 75-cm2 flasks and collected in 10 ml of cold 10× PBS. Cells were centrifuged at 8,000g at 4°C for 10 min. The pellet was resuspended in 500 μl phosphate buffer (100 mM KH2PO4+ 2 mM MgCl2, pH 7.4). The cell suspension was freeze-thawed (−80 to 37°C) three times and sonicated for 15 s in ice water. Heme oxygenase activity was then assayed as previously described (18).
Measurement of HO-1 mRNA by Northern blot analysis.
Cells were washed with cold PBS and then lysed directly in flasks by addition of guanidinium thiocyanate buffer, and total RNA was extracted using the method of Chomczynski and Sacchi (2). Total RNA (10 μg/lane) was run on a 1.3% denaturing agarose gel containing 2.2 M formaldehyde and transferred onto a nylon membrane, according to the method of Tyrrell and Basu Modak (35). The membrane was hybridized using [α-32P]dCTP-labeled cDNA probes to the rat HO-1 gene (the 88- to 971-nucleotide residue fragment of the rat heme oxygenase gene) (30) and the rat glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene. The hybridized membranes were exposed to radiographic film.
Measurement of HO-1 mRNA by RT-PCR.
RT-PCR was used to verify the Northern blot analysis results. Total RNA (1 μg) was reverse transcribed using random hexamer primers (GeneAmp RNA PCR kit, Perkin-Elmer, Norwalk, CT). With the use of a manual hot start, the cDNA was amplified using 2.5 units of AmpliTaq DNA polymerase (Perkin-Elmer), with the 3′ and 5′ primers for both β-actin and HO-1 being added to the same reaction tube (these yielded products of 247 and 583 bp, respectively). After an initial melting at 95°C, the PCR mixtures were amplified using a UNO II thermocycler (Biometra, Göttingen, Germany) for a total of 34 cycles, using a two-step protocol of melting at 95°C for 1 min and annealing at 61°C for 1.5 min. The product (20 μl/lane) was run on a 2.5% agarose gel (Metaphor agarose, FMC Bioproducts, Rockland, ME).
Measurement of HO-1 protein.
HO-1 protein was measured using Western blot analysis. Cells were manually scraped from 75-cm2flasks and collected in 10 ml of cold PBS. Cells were centrifuged at 8,000 g at 4°C for 10 min. The pellet was resuspended in 500 μl PBS containing 1% Triton X-100. The total amount of protein was measured using a commercial protein assay kit (Bio-Rad Laboratories, Hemel Hempstead, UK), and 30 μg of total protein were boiled in Laemmli buffer (10). Samples were separated on a 15% SDS-polyacrylamide resolving gel. Proteins were electrophoretically transferred onto a nitrocellulose membrane, and nonspecific antibody binding was blocked with 3% nonfat dried milk. The membrane was then probed with anti-HO-1 antibody (1:1,000 dilution) for 2 h at room temperature. Blots were visualized using an alkaline phosphatase staining kit (Rabbit ExtrAvidin, Sigma-Aldrich, Poole, UK).
Measurement of cell viability.
L6.G8 cells were seeded in 96-well plates at a density of 106 cells/ml and grown to confluence in complete medium. Incubations were performed as described in Cell culture and incubationabove. At the end of the experimental incubation period, the medium was replaced with tetrazolium reagent (CellTiter 96 AQueous One Solution Cell Proliferation Assay, Promega, Southampton, UK), diluted in complete medium according to the manufacturer’s protocol. Cells were then further incubated for 60 min at 37°C. The generation of the formazan product was assessed optically by measuring the absorbance at 490 nm using a plate reader and analytical software (Spectra Max250 and SOFTmax PRO, respectively, Molecular Devices, Crawley, UK). Results were expressed as a percentage of the control group.
One-way ANOVAs followed by Bonferronit-tests were used for the statistical analyses of data. All values were expressed as means ± SE, and differences between groups were considered significant atP < 0.05.
Effect of hemin and SNP concentration on heme oxygenase activity.
To examine whether hemin and SNP increased heme oxygenase activity, L6.G8 cells were treated for 6 h with incubation medium containing either hemin or SNP. Figure1 Aillustrates the change in heme oxygenase activity after incubation with a range of concentrations of hemin. Heme oxygenase activity was significantly elevated when cells were treated with 50 μM hemin (P < 0.01) and continued to increase in a concentration-dependent manner with a peak at 200 μM. At this concentration there was a 7.8-fold increase in heme oxygenase activity compared with the control value. At higher concentrations of hemin (300–400 μM), a slight but significant decrease from the maximum in heme oxygenase activity was observed (P < 0.05). A concentration-dependent response was also seen after incubation of cells with SNP (Fig. 1 B). Heme oxygenase activity significantly increased at a concentration of 0.1 mM SNP (P < 0.01), rising to a peak at 0.5 mM. At this concentration, there was a 5.0-fold increase in activity compared with the control value. Similarly to hemin treatment, exposure of cells to higher concentrations of SNP (1–2 mM) resulted in a significant decrease in activity from the maximum (P < 0.05).
Effect of duration of incubation with hemin or SNP on heme oxygenase activity.
To examine the rate of change of heme oxygenase activity in response to hemin and SNP, L6.G8 cells were treated with incubation medium for different durations (0–24 h). With the use of the optimal concentration of hemin (200 μM) and SNP (0.5 mM) for maximal heme oxygenase activation, heme oxygenase activity increased in a time-dependent manner compared with control (Fig.2, A andB). Within 3 h, heme oxygenase activity was significantly increased in response to both hemin and SNP stimulation (P < 0.01). Heme oxygenase activity continued to increase in a time-dependent manner with a peak at 6 h for both treatments. In both groups, heme oxygenase activity showed a decrease from maximum after a 24-h incubation, although it remained significantly greater than control (P < 0.05 with SNP andP < 0.01 with hemin).
Induction of HO-1 mRNA and protein by hemin and SNP.
To establish whether changes in heme oxygenase activity following hemin and SNP treatment were associated with changes in gene expression and de novo protein synthesis, HO-1 mRNA and protein levels were determined. HO-1 mRNA expression in L6.G8 cells following incubation with hemin was analyzed by Northern blot analysis (Fig.3 A). A concentration-dependent response in HO-1 mRNA levels was seen with a marked increase, compared with control, in HO-1 transcript across the entire concentration range (50–400 μM). These results were confirmed by RT-PCR using specific primers for HO-1 (Fig.3 B). Similarly, Western blot analysis of hemin-treated cells (Fig.3 C) showed a marked concentration-dependent increase in HO-1 protein levels above control, which paralleled the changes in HO-1 mRNA levels. It is interesting to note that HO-1 mRNA and protein levels increased during incubation with 0–200 μM hemin to concur with changes in heme oxygenase activity. At higher concentrations of 300 and 400 μM hemin, however, HO-1 mRNA and protein levels increased further, whereas heme oxygenase activity decreased from maximum. After incubation of L6.G8 cells with SNP, a marked increase in HO-1 mRNA was seen. Once again, HO-1 mRNA did not appear to diminish with higher concentrations (1 and 2 mM SNP), in contrast to changes seen in heme oxygenase activity (Fig.4).
Effect of HCB on SNP-stimulated heme oxygenase activity and HO-1 mRNA expression.
To determine whether heme oxygenase activity is stimulated by the release of NO from SNP, cells were incubated with HCB, an NO scavenger. The increase in heme oxygenase activity with 0.5 mM SNP was significantly (P < 0.01) attenuated by 0.5 mM HCB to 38% of the SNP-stimulated value. HCB alone had no effect on the basal level of heme oxygenase activity (Fig.5). These changes in heme oxygenase activity were reflected by changes in HO-1 gene expression. HO-1 mRNA expression as determined by Northern blot analysis is shown in Fig.6 A. Compared with control, a marked increase in HO-1 expression was seen in response to SNP and this was greatly attenuated by coincubation of cells with 1.0 mM HCB. To discriminate between the NO-scavenging properties and other free radical-scavenging properties of HCB, a further experiment was performed. Cells were incubated for 6 h in the presence of 10 μM cadmium chloride, with and without 1.0 mM HCB. HO-1 protein was greatly increased in cells incubated with SNP or cadmium alone. Simultaneous incubation with HCB markedly reduced both the amount of HO-1 mRNA and protein in SNP-treated cells but not in cadmium-treated cells (Fig. 6, A andB).
Effect of hemin and SNP on cell viability.
Hemin and SNP, through the release of iron and cyanide and the generation of free radicals, may be harmful to cells. To establish the conditions under which hemin and SNP cause significant cellular damage, cell viability was determined using a tetrazolium assay following incubation with various concentrations of hemin and SNP. As shown in Fig. 7 A, there was a gradual concentration-dependent decrease in viability, compared with control cells, which was more marked in the SNP- than the hemin-treated group. At 200 μM hemin and 0.5 mM SNP (both representing concentrations of maximal heme oxygenase activity), cell viability decreased to 89 ± 1.4% and 82 ± 2.1% of control, respectively. At the highest concentrations used (400 μM hemin and 2 mM SNP), the decrease in cell viability was most dramatic, declining to 75 ± 4.0% and 28 ± 1.0%, respectively. Cell viability also declined with duration of incubation (Fig.7 B). At 6 h, both hemin and SNP caused a significant decrease in cell viability (88 ± 1.5% and 84 ± 2.2%, respectively). An even greater reduction was seen at 24 h, resulting in cell viability of 77 ± 14% in hemin-treated cells and 45 ± 7.5% in SNP-treated cells, compared with control. The decline in heme oxygenase activity seen with both higher concentrations of inducing agent and longer durations of incubation coincided with the decrease in cell viability.
In this study, we have demonstrated that heme oxygenase activity can be stimulated by both hemin and SNP in L6.G8 skeletal myoblast cells and that this activation is associated with a concomitant increase in both HO-1 mRNA and protein levels. Our results support the finding of Essig et al. (4) that HO-1 can be stimulated in rat skeletal muscle. The present study suggests that heme and NO may be important regulators of HO-1 and heme turnover in skeletal muscle tissue in vivo. This could be of clinical relevance in certain pathophysiological situations, such as ischemia, which are characterized by heme release (23) and the cytokine-mediated increase in NO levels (21).
We have shown that the response to hemin stimulation in L6.G8 cells is very similar to that of other cell types. A significant increase in heme oxygenase activity has been found in human macrophages and human glioma cells after a 5-h incubation with hemin (37). Similarly, an increase was seen in pig alveolar macrophages with a peak at incubations of 5–7 h and a gradual decline in stimulated levels of heme oxygenase activity at longer incubation periods (31). These findings agree with our results, showing a peak in heme oxygenase activity after a 6-h incubation with a subsequent decline from maximum at longer durations. In both of these studies, lower concentrations of hemin were used than in the present study. Shibahara et al. (31) found a peak in heme oxygenase activity at 5 μM hemin, with a decline from maximum at higher concentrations, whereas L6.G8 cells responded maximally at 200 μM hemin but similarly declined at higher concentrations. L6.G8 cells may be less sensitive to hemin compared with other cell types studied or this difference may be a reflection of the incubation conditions. Our study was performed in serum-rich medium, whereas the other two studies were performed in serum-free medium. The added serum may reduce the amount of hemin available for cell stimulation because of binding to proteins such as albumin.
Western and Northern blot analyses (confirmed by RT-PCR) were performed to establish whether changes in heme oxygenase activity were a result of transcriptional induction and de novo protein synthesis or due to posttranslational protein activation. Both HO-1 protein and mRNA levels were found to increase with increasing hemin concentration, thereby confirming that changes in heme oxygenase activity occurred at the transcriptional level. No decreases in either protein or mRNA levels were observed at higher concentrations of hemin, indicating that the decline from maximum in heme oxygenase activity was not due to downregulation of gene expression. This suggests that at very high concentrations (300–400 μM) hemin remains an inducer of HO-1 but that it also can potentially inhibit the activity of the heme oxygenase enzyme.
The response of L6.G8 cells to SNP was also similar to that of other cell types studied. Bovine aortic endothelial cells showed a significant increase in HO-1 protein expression and heme oxygenase activity after incubations of 4 and 6 h with 1 mM SNP (5, 17). Similarly, rat vascular smooth muscle cells incubated with 1 mM SNP showed a significant increase in HO-1 protein expression after 4 h, which, however, continued to increase up to 24 h (3). In the present study, we also found that HO-1 protein levels increased in a concentration-dependent manner across the range of 0.1–1.0 mM SNP. L6.G8 cells responded to SNP with maximal heme oxygenase activity at 0.5 mM. In contrast to rat vascular smooth muscle cells, a decline from maximum in heme oxygenase activity was observed at higher concentrations (3). This suggests that L6.G8 skeletal muscle cells may be more sensitive than vascular smooth muscle cells to the potential toxic effect of SNP or its metabolites.
Both hemin and SNP caused loss of cell viability at all concentrations and durations examined. The data indicate that when both hemin and SNP induce heme oxygenase activity they also cause cellular damage. This damage may be caused by the inducers directly, by their metabolites, or via free radical formation. It is interesting to observe that at maximal heme oxygenase activity (6-h incubation with 200 μM hemin or 0.5 mM SNP) cell viability was similar with both hemin and SNP treatment, decreasing to 89 and 83% of control, respectively. With longer durations and higher concentrations of inducer, heme oxygenase activity diminished from its peak value. This decrease correlated directly with a further and more dramatic loss in cell viability. Despite this decrease in cell viability, both HO-1 mRNA and protein expression did not decline with higher concentrations of hemin (300 and 400 μM). This indicates that, under conditions of extreme stress, hemin-induced HO-1 expression is better preserved than enzyme function, suggesting that loss of heme oxygenase activity may correlate with pronounced impairment of cell viability. The possibility exists that at very high concentrations of inducer the HO-1 protein becomes a target for damage by free radicals generated by SNP or heme.
SNP is metabolized to a number of products, many of which could potentially be implicated in HO-1 induction. These include NO, iron, cyanide, and oxygen free radicals (26). HCB is a known scavenger of NO and was used to assess the contribution of NO to SNP-mediated increases in heme oxygenase activity. The attenuation of SNP-stimulated heme oxygenase activity by HCB in L6.G8 cells is consistent with findings in bovine aortic endothelial cells. Foresti et al. (5) showed a significant reduction, by about two-thirds, in SNP-stimulated heme oxygenase activity by 0.5 mM HCB (compared with a reduction of 62% found in this study). The fact that HCB did not completely suppress the increase in heme oxygenase activity and HO-1 mRNA expression is probably due to either incomplete blockade of NO or participation of other metabolites of SNP in HO-1 stimulation. Cyanide does not appear to be an inducer of HO-1 in vitro (17) and therefore was unlikely to contribute to the stimulation of heme oxygenase activity by SNP in our experiments. To exclude the contribution of free radicals, either generated from iron via the Fenton reaction or released directly from SNP degradation, additional experiments were conducted by exposing cells to cadmium. Cadmium is an extremely potent inducer of HO-1 (8) and is known to generate free radicals, particularly hydroxyl radicals (28). HCB did not reduce the degree of heme oxygenase induction by cadmium. This strongly suggests that, in our experiments, HCB is not a scavenger of free radicals but is a specific scavenger of NO, released from SNP. It is therefore unlikely that the SNP-stimulated heme oxygenase activity attenuated by HCB is mediated by free radicals other than NO or its derivatives (5). From these findings, it appears that, of all the potential inducers of HO-1 released from SNP, NO has by far the greatest contribution. This adds further supporting evidence to previously published data on the significance of NO in HO-1 induction (3, 5, 17).
The precise mechanism of HO-1 induction is not known. Many inducible genes are expressed in response to activation of various transcriptional factors by a variety of inducing agents. The binding sites of many transcriptional factors have been identified in the promoter region of the HO-1 gene, and it appears that HO-1 expression is regulated by the activation and binding of such transcriptional factors to these regions (1). An increase in the binding of a number of transcriptional factors in response to hemin treatment has been demonstrated, most significantly activator protein-2 and nuclear transcription factor-κB (34). Heme is a known potent inducer of HO-1, and, if it stimulates HO-1 induction via activation of transcriptional factors, then modulation of intracellular free heme concentration would be a sensitive mechanism for HO-1 regulation. The mechanism of NO induction of HO-1 is less clear because no transcriptional factor sensitive to NO has been identified. NO, however, does appear to be able to displace heme from heme proteins (9); therefore, it is possible that NO is capable of regulating HO-1 levels via modulation of the intracellular free heme concentration.
In summary, we have been able to demonstrate that heme oxygenase activity is significantly increased in rat skeletal myoblasts following exposure to hemin and the NO donor, SNP, and that changes in activity are paralleled by both increased HO-1 protein levels and mRNA expression. The role of heme oxygenase in skeletal muscle is still unclear. Its two functions of cellular protection against oxidative stress and regulation of heme turnover would suggest an important biological role in skeletal muscle cells under both normal and pathophysiological conditions. The inducible isoform, HO-1, has been recently identified in rat skeletal muscle tissue in vivo (4). We have been able to verify significant induction of HO-1 by ischemia-reperfusion in human skeletal muscle samples taken from free muscle flap transfers before and during vessel division and after reanastomosis (data not shown). It has been demonstrated that HO-1 is protective against heme-mediated damage in the kidney during rhabdomyolysis following muscle ischemia (20). On the basis of the evidence in skeletal myoblast cells presented here, the possibility exists that HO-1 may have a similar function in heme-rich tissues, such as muscle. Heme, through its ability both to release iron for use in the generation of the hydroxyl radical and to induce the stress protein HO-1, has a pivotal role in the balance between cellular damage and protection during the breakdown of heme proteins in muscle ischemia. The regulation of heme turnover is critical to this; therefore, the heme oxygenase proteins, especially HO-1, can be expected to contribute significantly to cellular protection.
We are grateful to Prof. S. Shibahara, Tohoku University School of Medicine, Sendai, Japan, for donating the cDNA probe for the rat HO-1 gene; to Dr. S. Mohammed, Royal Free Hospital Medical School, London, UK, for donating the cDNA probe for the rat GAPDH gene; and also to Dr. R. Martin, Blond McIndoe Centre, Queen Victoria Hospital, East Grinstead, Sussex, UK, for help in the preparation of the cDNA probe to the HO-1 gene.
Address for reprint requests: M. Vesely or R. Motterlini, Vascular Biology Unit, Dept. of Surgical Research, Y Block, NPIMR, Northwick Park Hospital, Watford Rd., Harrow, Middlesex HA1 3UJ, UK.
This work was supported in part by Restoration of Appearance and Functional Trust Institute of Plastic Surgery.
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- Copyright © 1998 the American Physiological Society