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1 Division of Pulmonary and Critical Care Medicine, Department of Medicine, and 3 Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44109; and 2 Department of Medicine, Medical College of Georgia, Augusta, Georgia 30912
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ABSTRACT |
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 TOP
 ABSTRACT
 INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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Reactive oxygen
species contribute to diaphragm dysfunction in certain
pathophysiological conditions (i.e., sepsis and fatigue). However, the precise alterations induced by reactive oxygen species or
the specific species that are responsible for the derangements in
skeletal muscle function are incompletely understood. In this study, we
evaluated the effect of the superoxide anion radical (O2
·), hydroxyl radical (·OH), and hydrogen
peroxide (H2O2) on maximum calcium-activated
force (Fmax) and calcium sensitivity of the contractile
apparatus in chemically skinned (Triton X-100) single rat diaphragm
fibers. O2· was generated using the
xanthine/xanthine oxidase system; ·OH was generated using 1 mM
FeCl2, 1 mM ascorbate, and 1 mM
H2O2; and H2O2 was
added directly to the bathing medium. Exposure to O2· or ·OH significantly decreased
Fmax by 14.5% (P < 0.05) and 43.9%
(P < 0.005), respectively. ·OH had no effect on
Ca2+ sensitivity. Neither 10 nor 1,000 µM
H2O2 significantly altered Fmax or
Ca2+ sensitivity. We conclude that the diaphragm is
susceptible to alterations induced by a direct effect of ·OH and
O2·, but not H2O2, on the
contractile proteins, which could, in part, be responsible for
prolonged depression in contractility associated with respiratory
muscle dysfunction in certain pathophysiological conditions.
free radicals; reactive oxygen species; skinned muscle fibers; respiratory muscle; skeletal muscle
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INTRODUCTION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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THE DIAPHRAGM
GENERATES oxygen-derived free radical species at rest and during
contraction (12, 21, 37). Several of the most important of
these reactive oxygen species (ROS) include hydrogen peroxide
(H2O2), the superoxide anion radical
(O2·), and the hydroxyl radical (·OH). Whereas
low levels of these intermediates are thought to be necessary for
optimal function of skeletal muscles under normal conditions
(36), in pathological states, including sepsis (6,
16) and ischemia-reperfusion injury (46) and
during fatigue (5, 21), it has been postulated that
increased oxidative stress leads to tissue injury and, consequently, skeletal muscle dysfunction (4, 41). Measurements of
increased levels of lipid peroxidation by-products (3, 44)
and the observation that administration of selective or nonselective
free radical scavengers partially ameliorates muscle dysfunction
provide indirect evidence that enhanced ROS generation is an important factor in skeletal muscle dysfunction in a variety of conditions (11, 39, 40, 43). More direct evidence for the toxicity of
ROS has been demonstrated by infusion of a free radical-generating solution into the diaphragm of dogs, which resulted in decreased force
generation and a shift in the force-frequency relationship downward and
to the right (31).
The precise alterations induced by ROS or the specific species that are
responsible for the derangements in skeletal muscle function are
incompletely understood. Whereas some studies have evaluated the
effects of free radicals on skeletal muscle sarcoplasmic reticulum (SR)
and mitochondria (13, 35), only limited work has been
performed to determine the effects of ROS on skeletal muscle
contractile proteins. Moreover, most of the reports that have examined
this issue in skeletal muscle have studied the effects produced by
exposure of intact muscle fibers to exogenous free radical-generating
solutions (2). These previous experiments have fundamental
limitations, because indirect metabolic effects resulting from the
actions of free radicals on intermediary metabolic pathways may well
have been responsible for all of the phenomena observed.
Methodologically, a more direct approach to this problem is to
determine the effects of free radical-generating solutions on
"skinned" muscle fibers (i.e., single fibers in which the
sarcolemma, SR, and mitochondria have been removed, thus permitting
direct functional assessment and access to the contractile proteins). Only a few reports have directly evaluated the effects of free radical
species on contractile protein function in skeletal muscle using
skinned fibers, and only the effects of nitric oxide, peroxynitrite, and H2O2 have been examined (8, 32,
45). Importantly, no study has explicitly examined the direct
effects of ·OH or O2· on contractile protein
function in any skeletal muscle using skinned fibers. It is known that
different free radical species possess different propensities to
chemically modify cellular constituents. For example, peroxynitrite and
nitric oxide produce nitrosylation of aromatic amino acid side chains,
whereas superoxide and hydroxyl anions do not. Hydroxyl and
peroxynitrite induce lipid peroxidation, whereas superoxide and nitric
oxide do not (19). As a result, it is possible that
individual free radical species may produce qualitatively and
quantitatively different effects on contractile protein function, and
it is necessary to study each free radical species individually to
obtain a comprehensive understanding of the potential functional
alterations of ROS on skeletal muscle.
The purpose of present study, therefore, was to examine the direct
effects of O2· and ·OH on the contractile
apparatus in chemically skinned, single skeletal muscle fibers. Because
it was necessary to utilize H2O2 as a component
of our ·OH generating solution, we also determined the effects of
H2O2 per se in our experimental system. We
chose to study fibers isolated from the diaphragm rather than limb
skeletal muscle, both because of the physiological importance of the
diaphragm (this is the only skeletal muscle whose function is critical
to sustain life) and because a number of studies have identified the
diaphragm as a target of free radical-mediated injury in several pathophysiological states. We specifically investigated the direct effects of the three free radical species studied on maximum
calcium-activated force (Fmax) of the contractile proteins
and, when possible, also determined the effects of these species on the
calcium (Ca2+) sensitivity of the contractile apparatus.
Because it is known that ·OH are more chemically reactive with
proteins, we hypothesized that ·OH would produce greater alterations
in contractile protein function than either O2· or
H2O2 (33). Moreover, because one
previous study has shown that calcium binding to isolated skeletal
muscle troponin C is reduced by exposure to free radical-generating
solutions, we postulated that ·OH and O2· would
reduce calcium sensitivity of the contractile proteins, shifting the
force vs. pCa relationship to the right (18).
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METHODS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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General Approach
All experiments were conducted on single diaphragm fibers removed from adult Sprague-Dawley rats (all weighing <200 g), which were killed by cervical dislocation. This study was approved by the Medical College of Georgia Institutional Animal Care and Use Committee, and all procedures were performed in accordance with experimental guidelines for that institution and in accordance with American Physiological Society guidelines. After death, the diaphragm was carefully detached from its intercostal insertions and placed in a dissecting dish containing a relaxing solution with the following composition (in mM): 1.0 Mg2+, 5.0 MgATP, 15 phosphocreatine, 140.0 potassium methanesulfonate, 50.0 imidazole, and 10.0 EGTA, with pCa >8.5 and pH 7.0. Ionic strength was adjusted to 200. This solution also contained the following protease inhibitors to protect the fibers from the damaging effects of proteolysis: 0.1 mM phenylmethysulfonyl fluoride, 0.1 mM leupeptin, 1.0 mM benzamidine, 10 µM aprotinin, and 1 mM dithiothreitol (DTT).The diaphragm was subsequently divided into small strips, and some
bundles were stored at 
20°C in relaxing solution containing 50%
glycerol and protease inhibitors. In this storage solution, CTP was
used in place of ATP to prevent phosphorylation of myosin light chains.
All single-fiber assessments were completed within 48-72 h of dissection.
On the day that fiber characteristics were assessed, diaphragm strips were removed from storage solution, placed in DTT-free relaxing solution (i.e., all components were the same as listed in the previous paragraph except that DTT was not included), and allowed to warm to room temperature. Small bundles of ~10 fibers were then separated from the whole muscle by gently pulling on one end of the muscle with a pair of fine-tipped forceps while the other end of the muscle was held stationary with a second pair of forceps. Fiber bundles were immersed for 30 min in 0.1% Triton X-100, an ionic detergent that eliminates the membranes of the sarcolemma, SR, and mitochondria, leaving only the contractile proteins intact.
After incubation, bundles were removed from Triton X-100 and placed in relaxing solution, and individual fibers were teased from the muscle bundles. Single fibers were then mounted between an optoelectric force transducer (Scientific Instruments, Heidelberg, Germany) and a movable arm by wrapping the ends of each fiber around stainless steel wires. Fiber length was adjusted to achieve a resting sarcomere length of 2.6 µm as indicated by its helium-neon laser diffraction pattern. Length remained constant throughout the experiments. The cross-sectional area of each fiber was determined after sarcomere length was adjusted, by measuring the diameter of the fiber using a micrometer attached to the eyepiece of the microscope binocular. Area was calculated, assuming a cylindrical shape for the fiber.
Force vs. pCa curves were constructed for fibers under baseline conditions by immersing them in solutions of increasing calcium concentrations and recording tension on a strip recorder. Once peak tension was achieved in a given solution, fibers were rapidly switched to the next solution by means of a spring-loaded Plexiglas tray. The composition of all solutions used in this study was calculated by using a computer program (Borland International, Scotts Valley, CA) that takes into account stability constants and stock solutions to produce final solutions of the correct ionic strength and pCa (12). Specifically, the solutions used to examine the force vs. pCa relationship were of the following composition (in mM): 1.0 Mg2+, 1.0 MgATP, 15 phosphocreatine, 110.0 potassium methanesulfonate, 20.0 imidazole, and 5.0 EGTA, pH 7.0. Ionic strength was 200. Ca2+ was added to yield solutions of the desired pCa.
The general experimental protocol consisted of the following steps. 1) Fibers were submerged in a solution containing no added calcium (pCa 8.5), followed by sequential exposure to 11 different calcium solutions, namely pCa 6.0, 5.90, 5.80, 5.70, 5.60, 5.50, 5.40, 5.30, 5.20, 5.0, and 4.0, to establish the baseline force vs. pCa relationship. 2) Fibers were relaxed in pCa 8.5 solution and then transferred to another trough containing pCa 8.5, where they were exposed to the ROS-generating solution or individual components of the ROS-generating solutions. 3) After ROS exposure, a second force vs. pCa curve was generated. Modifications to the above protocol are outlined specifically below. All experiments were performed at room temperature, which averaged 22°C.
Experimental Procedures
We conducted three groups of experiments to determine the effect of each individual oxygen-derived species being investigated on Fmax and the calcium sensitivity in single, chemically skinned diaphragm fibers from adult animals. Groups 1, 2, and 3 consisted of fibers exposed to H2O2, O2·, or ·OH,
respectively. Details of the specific protocols for each group of
experiments are described below.
Protocol 1: Exposure to H2O2. To achieve exposure to H2O2 in single, skinned diaphragm fibers, either 10 or 1,000 µM H2O2 were added directly to the pCa 8.5 solution using a diluted standard 30% stock solution (Sigma Chemical, St. Louis, MO) to achieve the final desired concentration. The concentrations of the diluted H2O2 solutions were directly measured by spectrophotometer at 230 nm, assuming a molar extinction coefficient of 0.071 × 103 M/cm (9). We chose to evaluate two concentrations of H2O2; both doses have been commonly used in previous studies that evaluated the effects of H2O2 (7, 8, 10, 25, 26).
The protocol for exposure of single fibers to H2O2 consisted of steps 1-3 as outlined above; in step 2, the fibers were exposed for 5 min in pCa 8.5 to either 10 or 1,000 µM H2O2. In a separate group of in-time control fibers, H2O2 was not added to the solution in step 2.Protocol 2: Exposure to superoxide.
In the second series of experiments, the xanthine-xanthine oxidase
system was used to generate O2·. Before initiation
of the skinned fiber protocol, O2· generation with
50 µM xanthine and 0.91 mU xanthine oxidase in the pCa 8.5 solution
was confirmed spectrophotometrically by measuring the reduction of 0.05 mM cytochrome c at 550 nm. To confirm that the reactive
species was indeed O2·, superoxide dismutase (SOD;
15 µg/ml) was added to the above O2· generating
system. The amount of O2·, as determined by the
amount of SOD-inhibitable cytochrome c reduction, was
calculated based on the changes in the spectrophotometric readings at
550 nm in the presence and absence of SOD, assuming a molar extinction
coefficient of 18.5 × 103 M/cm.
O2· was produced in our system at a rate of 0.26 nmol · ml1 · min1 and
remained at this level for at least 10 min. The absence of ·OH in
this system was confirmed by using the salicylate trapping method
(12). For these latter determinations, 50 µM xanthine and 0.91 mU xanthine oxidase in pCa 8.5 solution were incubated with 1 mM sodium salicylate for 10 min (n = 3 determinations). Samples from the reaction mixture were assayed using HPLC for determination of 2,3- or 2,5-dihydroxybenzoic acid peaks
(12). We found no evidence of 2,3- or 2,5-dihydroxybenzoic
acid formation in any sample using this approach. This concentration of
O2· is lower than the maximum reported rates of
mitochondrial O2· generation (1.2 nmol · min1 · mg protein1)
(14) but is comparable to that which has previously been
used to evaluate the effects of O2· on the
contractile properties of skinned cardiac fibers (24). Additionally, we have previously shown that, during a fatiguing stimulation, there is extracellular release of O2·
at a rate of 0.70 ± 0.17 nmol/min in whole diaphragm preparations (21).
· generation. However, high concentrations of
O2· and an exposure time of 5 min made the fibers
extremely fragile, such that we were unable to evaluate the entire
force vs. pCa relationship for this series of experiments. Because the
individual components of the O2· generating system
could be responsible for any observed effects on Fmax, we
controlled for this possibility.
In these experiments, the protocol consisted of the following steps.
1) baseline Fmax (pCa 5.3) was obtained in all
fibers, followed by relaxation in pCa 8.5. 2) Fibers were
then exposed for 2 min in pCa 8.5 to which one of the following was
added: xanthine, xanthine oxidase, SOD, xanthine + xanthine
oxidase, or xanthine + xanthine oxidase + SOD. 3)
After exposure, a repeat measurement of Fmax in pCa 5.3 was
performed in all fibers. In-time control fibers were carried through
the same protocol but without exposure to O2· or
any component of the generating system.
Protocol 3: Exposure to ·OH. In the final series of experiments, ·OH was generated in pCa 8.5 using 1 mM FeCl2, 1 mM ascorbic acid, and 1,000 µM H2O2. Because our normal pCa 8.5 solution contains a significant amount of EGTA (5 mM), the solution used in these experiments had to be modified by reducing the EGTA to 0.05 mM to prevent chelation of the iron necessary to generate ·OH. Production of ·OH was confirmed by HPLC using the salicylate trapping method, as previously described (12). HPLC analysis showed that 31 µmol of 2,3-dihydroxybenzoic acid and 2,5-dihydroxybenzoic acid was produced in this system over a 5-min period.
The protocol used to assess the effects of ·OH and each individual component of the ·OH generating system on the force vs. pCa relationship was similar to protocol 2. In step 2, fibers were relaxed in pCa 8.5 (5 mM EGTA) and subsequently exposed for 5 min in pCa 8.5 (0.05 mM EGTA) to either 1 mM FeCl2, 1 mM FeCl2 + 1 mM ascorbic acid, or 1 mM FeCl2 + 1 mM ascorbic acid + 1,000 µM H2O2. Because we had already performed experiments to examine the effects of exposure to 1,000 µM H2O2 as well as run-down in the control fibers after 5 min (see Protocol 1: Exposure to H2O2), we used the results from these experiments in the present protocol. Furthermore, because no significant changes in Fmax were seen with a 5-min exposure to ascorbic acid in a series of preliminary experiments, full force vs. pCa curves were not performed for this component of the ·OH generating system.Fiber Typing
After each experiment, fibers were stored in SDS buffer solution containing 0.125 M Tris, 4% SDS, 20% glycerol, and 20 mM DTT at40°C. Fibers were analyzed for slow myosin heavy chain (MHC) using
Western Blot analysis. Samples were denatured at 95°C, and SDS-PAGE
was conducted under reducing conditions on a 6% separation gel with a
4% stacking gel. Proteins were transferred to a nylon membrane by
electroblotting (BioRad Immun-Lite Assay kit). The blot was blocked
overnight in a 5% nonfat milk powder dissolved in Tris-buffered saline
(500 mM NaCl, 20 mM Tris · HCl, pH 7.5) solution and
washed for 1 h in Tris-buffered saline + 0.05% Tween 20 (TTBS). The blot was incubated with a 1:50 diluted
monoclonal anti-MHC slow antibody (Accurate Chemical & Scientific,
Westbury, NY) in TTBS containing 1% nonfat milk powder and 0.1%
sodium azide for 4 h at room temperature. After immunoreaction,
the membrane was stored at 4°C overnight in the same solution. The
next day, the membrane was washed for 1 h in TTBS. Immunodetection
of the primary antibody against the anti-MHC slow antibody was carried out with a 1:3,000 diluted anti-mouse-horseradish peroxidase secondary antibody solution (TTBS with 1% nonfat milk powder and 0.1% sodium azide) for 2 h at room temperature. The membrane was washed again for 1 h, incubated with chemiluminescent detection reagent for 5 min, and exposed to an X-OMAT AR X-ray film for 5-10 min. Rabbit soleus muscle (10 µg total protein) was used as a control. The presence of the slow MHC classified the fiber type as slow. Of the 86 fibers used in this study, only two were identified as slow fibers, and
these were excluded from the data analysis.
Data Analysis and Statistics
SigmaPlot software (version 2.0, Jandel Scientific) was used to determine the constant N related to the steepness of the force vs. pCa relationship (N is a measure of the extent of cooperativity among the thin filaments) and the calcium concentration required for half-maximal activation (Ca50) (K) values for the force-pCa relationships from a best fit of the data to the modified Hill equation
![%Maximum force<IT>=100</IT>[Ca<SUP>2+</SUP>]<SUP><IT>N</IT></SUP><IT>/</IT>{(K)<SUP><IT>N</IT></SUP><IT>+</IT>[Ca<SUP>2+</SUP>]<SUP><IT>N</IT></SUP>}](/content/vol90/issue1/fulltext/45/img001.gif)
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·, and ·OH, or
one of the components of the generating system. The significance of the
effects of exposure to H2O2,
O2·, and ·OH, or to any component of one of the
generating systems in each individual fiber, was calculated using the
paired t-test (SigmaStat, version 2.0, Jandel Scientific).
The effects of each condition on groups of fibers utilized in
protocols 1, 2, and 3 were compared
with time-controlled fibers using one-way ANOVA, followed by post hoc
comparisons using the Student-Newman-Keuls all-pairwise multiple
comparison procedure. Because the individual components of the ROS
generating systems induced alterations in the contractile proteins, the
differences due to the specific ROS being studied in protocols
1, 2, and 3 were assessed by comparing the
results of ROS-exposed fibers to their appropriate control fibers. All
data were corrected for any direct effect of the individual components
of the generating systems on the contractile apparatus. A P
value < 0.05 was considered to be statistically significant. All
fast fibers studied were included in the data analysis.
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RESULTS |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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A total of 84 fast fibers was used in this study. Absolute
Fmax under baseline conditions (i.e., before exposure to
any of the three specific ROS being examined) averaged 126.2 ± 8.1 kPa. Cross-sectional area of the fibers averaged 4.29 ± 0.22 × 109 m2. There were no
significant differences in absolute force or cross-sectional area in
any of the different experimental groups under baseline conditions.
Group 1: Response to 10 or 1,000 µM H2O2 Exposure
Exposure to 10 or 1,000 µM H2O2 caused no significant decrease in Fmax compared with in-time control fibers (5 min in pCa 8.5 without H2O2). Specifically, Fmax decreased to 91.9 ± 1.0 and 90.3 ± 1.7% of initial baseline force in fibers treated with 10 µM H2O2 and in the control fibers, respectively (Fig. 1A). Similar results were obtained in fibers exposed to 1,000 µM H2O2 (94.4 ± 1.2% of initial baseline force). When we examined the effects of H2O2 exposure on the calcium sensitivity of the contractile apparatus, there were no significant changes in the Ca50 in fibers treated with 10 µM H2O2 (see Fig. 1B) or 1,000 µM H2O2 or in the in-time control fibers. The difference in the Ca50 after 5 min averaged 0.11 ± 0.05 µM Ca2+ in fibers treated with 10 µM H2O2, 0.13 ± 0.09 µM Ca2+ in fibers treated with 1,000 µM H2O2, and 0.22 ± 0.06 µM Ca2+ in the in-time control fibers. Furthermore, analysis of the Hill coefficient also showed no significant differences among groups. After 5 min, N values averaged 6.00 ± 0.40 in fibers treated with 10 µM H2O2, 4.56 ± 0.25 in fibers treated with 1,000 µM H2O2, and 5.04 ± 0.40 in the in-time control fibers. These results demonstrate that exposures to 10 or 1,000 µM H2O2 had no significant effect on Fmax or the calcium sensitivity (Ca50) of the contractile apparatus of single diaphragm fibers.
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Group 2: Response to O2· Exposure
·, or 50 µM xanthine with 0.91 mU xanthine oxidase and 15 µg/ml SOD. After
exposure, a second measurement of Fmax in pCa 5.3 was performed in all fibers. These results are shown in Fig.
2. Exposure to O2·
reduced force to 64.7 ± 2.3% of initial baseline force
(P < 0.001) in the same fiber. Compared with the
appropriate controls (i.e., xanthine + xanthine oxidase + SOD), the reduction in force after a 2-min exposure to
O2· represents a 14.5% decline, which is
significant (P < 0.05). This indicates that
O2· significantly alters the contractile properties
of single diaphragm fibers by inhibiting Fmax. After 2 min,
Fmax for the in-time control fibers was 94.1 ± 1.4%
of initial baseline force. In fibers exposed to either xanthine or SOD
for 2 min, Fmax was 96.8 ± 1.9 and 98.1 ± 1.7%, respectively, of the initial baseline force, which is not
significant. However, after exposure to xanthine oxidase alone, force
was 75.8 ± 3.1% of initial baseline force (P < 0.001). In fibers treated with xanthine + xanthine oxidase + SOD, force diminished to 75.7 ± 3.1% of initial baseline force
(P < 0.001). These reductions in Fmax are
almost identical and suggest that xanthine oxidase has a nonspecific
inhibitory effect on force generation.
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Group 3: Response to ·OH Exposure
In these experiments, an initial force vs. pCa relationship was determined for fibers under baseline conditions, and fibers were then exposed to either 1 mM FeCl2, 1 mM FeCl2 with 1 mM ascorbic acid, or 1 mM FeCl2 with 1 mM ascorbic acid and 1,000 µM H2O2 (to generate ·OH) for 5 min in pCa 8.5. After the 5-min exposure, a second force vs. pCa relationship was determined. In preliminary experiments, a 5-min exposure to 1 mM ascorbic acid in pCa 8.5 produced no significant decrease in Fmax compared with control fibers (93.3 ± 2.7 vs. 90.3 ± 1.7% in controls). Furthermore, as demonstrated above in Group 1: Response to 10 or 1,000 µM H2O2 Exposure, 1,000 µM H2O2 has no significant effect on either Fmax or Ca50.When we examined the effects of the other components of the ·OH
generating system and of ·OH per se, we found significant changes in
Fmax (Fig. 3). Specifically,
exposure to FeCl2 reduced force to 83.8 ± 7.9% of
the initial force, and exposure to FeCl2 with ascorbic acid
reduced force further to 68.6 ± 6.7% of initial baseline force.
However, when FeCl2, ascorbic acid, and
H2O2 were used to generate ·OH,
Fmax declined to 38.5 ± 5.0% of initial baseline
values (P < 0.0001). Compared with the appropriate
controls (FeCl2 with ascorbic acid), exposure to ·OH
resulted in a decline in force of 43.9% (P = 0.005).
The effects of FeCl2 and FeCl2 with ascorbic
acid on the force vs. pCa relationship are shown in Fig.
4, and those of ·OH are shown in Fig.
5.
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When we examined the effects of ·OH exposure or the individual components of the generating system (FeCl2 or FeCl2 with ascorbic acid) on the calcium sensitivity of the contractile apparatus, there were no significant differences observed among individual fibers or among groups. The Ca50 averaged 1.74 ± 0.05, 1.91 ± 0.10, and 1.90 ± 0.10 µM Ca2+ in fibers after exposure to FeCl2, FeCl2 with ascorbic acid, and ·OH, respectively. Furthermore, analysis of the Hill coefficient also showed no significant differences among groups. These data indicate that ·OH exposure significantly decreases Fmax but does not alter calcium sensitivity of the contractile proteins in single diaphragm fibers (Fig. 5).
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DISCUSSION |
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
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To our knowledge, this is the first study to examine the direct
effects of O2· and ·OH on the contractile
apparatus in single Triton-skinned skeletal muscle fibers. Exposure to
either O2· or ·OH resulted in significant
reductions in Fmax. We also found that the calcium
sensitivity of the contractile proteins was not altered with exposure
to ·OH. In contrast, H2O2 had no significant effect on Fmax or the calcium sensitivity of the
contractile apparatus.
In keeping with our initial hypothesis, ·OH had a more pronounced
effect on depressing Fmax than did either
O2· or H2O2. Our hypothesis
that calcium sensitivity would be altered is not supported by our
findings, because neither ·OH nor H2O2 altered the Ca50. These data provide a potential mechanism
by which O2· and ·OH could contribute to
respiratory muscle dysfunction via direct effects on the contractile apparatus.
Methodological Considerations
We employed a large number of control groups to assess the individual effects of components of the free radical-generating solutions used in this study. By doing so, we were able to separate the nonspecific effects produced by these components from the specific effects of O2· and ·OH on force generation.
Surprisingly, we found that the nonspecific effects produced by several
components of the generating systems were quite pronounced (e.g.,
xanthine oxidase produced a 24.2% reduction in Fmax). This
raises the question as to whether or not these nonspecific effects may
have altered the contractile proteins in such a way as to artifactually
heighten the depressant effects of O2· and ·OH.
For example, it is conceivable that one of these chemical components
might induce alterations in protein conformation and expose an
oxidant-sensitive site that might not normally be susceptible to
redox-mediated modification. There are, however, no studies in the
literature that suggest that this might occur. Moreover, some data
indicate that oxidant-sensitive sites are easily accessible to ROS when
contractile proteins are in their native configuration. For example,
Perkins et al. (32) have suggested that sulfhydryl groups
within the actomyosin ATPase are readily accessible and exquisitely
sensitive to redox modification. Therefore, whereas we cannot entirely
exclude the possibility that some artifact may have magnified the
depressant effects of O2· and ·OH on the
contractile proteins, we think such a phenomenon is unlikely.
Even though our experiments were designed in such a way that we
attempted to examine the effects of O2·, ·OH, and
H2O2 individually on the contractile proteins,
it must be understood that these chemical species are interrelated and
solutions of any one of these may have been contaminated with the other
radical species. Our findings, however, would argue that such potential
contamination is of trivial importance. For example,
H2O2 had no effect on Fmax,
O2· had small effects on Fmax, and
·OH had large effects on Fmax. It is not possible that
significant amounts of O2· or ·OH were present in
the H2O2 generating solution, because such an
eventuality should have resulted in a significant effect of
H2O2 on force generation, which was not seen.
Conversely, H2O2 alone had no effect on force
generation; therefore, the presence of H2O2
could not have affected the alterations seen with the O2· or ·OH. Furthermore, we excluded the presence
of ·OH in the O2· generating solution (see
METHODS). Nevertheless, there are no waterproof barriers
between free radical species, and it is even possible that a variety of
uncharacterized low-molecular-weight radical species may have been
generated in the complex, biological milieu studied in the present
experiments. However, in vivo responses to ROS are likely to involve
the participation of such daughter reaction species. As a result, we
would still argue that the physiological responses demonstrated in the
present study represent the overall effects produced when the
contractile proteins are exposed to O2·, ·OH, or
H2O2.
DTT, a reducing agent, was selectively used in some of the solutions employed in these experiments. The only solutions containing DTT were those used to store or to dissect fibers. This chemical was not included in any of the solutions actually used to construct force vs. pCa relationships; i.e., this agent was not included in any of the activating solutions used in this study. DTT was also not included in the pCa 8.5 solution, nor was it included in any of the free radical-generating solutions. It is, therefore, extremely unlikely that the presence of DTT had any significant effects on the results obtained in our studies.
In the present study, we only present data for fast fibers in the
diaphragm. Moreover, the technique we used for fiber typing did not
distinguish between subtypes of fast fibers. However, we observed an
extremely uniform response to the various ROS being tested. It seems
unlikely, therefore, that there is significant subtype-dependent
variation in the contractile protein response to
O2· or ·OH.
Comparison to Previous Studies
Our findings in skinned diaphragm fibers exposed to H2O2 confirm those previously reported in the literature. Brotto and Nosek (8) examined the effects of a 5-min exposure to 1,000 µM H2O2 on the contractile apparatus in single skinned fibers from the extensor digitorum longus of rats and found no changes in Fmax or calcium sensitivity. Similar studies in Triton-skinned cardiac myocytes using 10 mM H2O2 and exposure times of up to 60 min (25) showed that there were no significant changes in myofibrillar force generation or calcium sensitivity, leading the authors to conclude that H2O2 was not an important modulator of the contractile proteins per se, even under pathophysiological conditions (25).Andrade et al. (1) examined the effects of exogenously administered H2O2 on force and myoplasmic calcium concentrations ([Ca2+]i, where i is intracellular) in single, intact fibers from the flexor brevis muscle of mice and found that, at more physiological concentrations (150-300 µM H2O2), H2O2 effects were biphasic. With short exposure times, submaximal tetanic [Ca2+]i was unchanged, but force generation actually increased. With longer exposures, force declined and was independent of changes in submaximal tetanic [Ca2+]i. The authors suggest that contractile protein function in intact, single skeletal muscle fibers is sensitive to changes in cellular redox status, with the potential for biphasic responses (1). It is important to note, however, that, in the intact single-fiber preparation, the observed effects from exogenously administered H2O2 could be due to alterations at a myriad of sites within the myocyte (i.e., SR, pH, metabolism, etc.) or to effects of H2O2 derivatives. It is also possible that the observed effects are due to more than one modification in the cellular machinery, producing changes where physiological consequences are functionally opposing. In the present study, we have eliminated many of these confounding factors by studying single Triton-skinned fibers. In our experiments, potential alterations in the sarcolemma, the SR, the mitochondria, the membranes of other subcellular organelles, or cytoplasmic enzymes by ROS can be ignored, because this preparation contains only the contractile proteins. Therefore, based on our results, we conclude that the contractile proteins per se are not directly modified by exposure to H2O2.
To our knowledge, our studies are the first to examine the direct
effects of O2· exposure on contractile function in
single skinned skeletal muscle fibers and, specifically, in the
diaphragm. MacFarlane and Miller (24) previously examined
the direct effects of O2· on skinned cardiac
myocytes and found that the contractile apparatus was extremely
sensitive to this particular reactive oxygen intermediate. In those
studies, exposure to as little as 2 mU/ml of xanthine oxidase for only
2 min markedly diminished absolute force generation to levels <20% of
the initial preexposure force. These effects on force were both
concentration and time dependent. Furthermore, despite changes in
force, there were no changes in calcium sensitivity (24).
Although the amount of O2· generated in our system
was considerably lower than that used by MacFarlane and Miller, our
findings in the diaphragm parallel these findings in cardiac muscle
with regard to decrements in maximal force generation. We were not able
to examine the force vs. pCa relationship in the
O2· experiments and, therefore, are unable to make
any conclusions with regard to the effects of O2·
exposure on the calcium sensitivity of the contractile apparatus.
The present findings demonstrate that, when skeletal muscle contractile proteins are exposed to the ·OH, Fmax decreases significantly without producing changes in calcium sensitivity. We are not aware of any other studies in skeletal muscle that have examined the direct effect of ·OH on the force-generating capacity of the contractile proteins. However, studies that employed spin traps in Triton skinned rabbit psoas muscle exposed to a ·OH generating system showed that ·OH can specifically modify the Cys-707 residue of the SH-1 group of myosin (22). In other studies, using in vivo iron overload as a model of oxidative stress, skeletal muscle actin and myosin have been shown to be oxidatively modified (30). Although these studies shed light on potential sites of protein modification by ·OH, the functional consequences of these modifications in skeletal muscle were not examined in these previous studies. Our results confirm that ·OH is a potent reactive oxygen intermediate that is able to alter the contractile proteins significantly.
Potential Sites of Free Radical-Induced Alterations of the Contractile Proteins
The specific site or sites of alteration in the contractile proteins induced by exposure to the different free radical-generating solutions used in the present study are not known. Potential targets include oxidation of critical thiol residues on myosin, actin, troponin, or tropomyosin. Other possible mechanisms include free radical-mediated biochemical modifications that induce changes in the tertiary structures of these proteins. Free radical-induced alterations in tertiary structure of proteins are well recognized as the major mechanism underlying protein degradation by the proteosome complex. It is quite possible that similar alterations in the contractile proteins could modify protein-protein contact, disrupting interactions among myosin, actin, troponin, and tropomyosin.One of the potential sites of alteration by the ROS examined in the
present study is myosin. In skinned cardiac myocytes, MacFarlane and
Miller (24) found that the contractile apparatus was
extremely sensitive to O2· and that these effects
were both concentration and time dependent. Despite significant
alterations in force, there were no changes in Ca2+
sensitivity. However, during rigor, where there is no active cross-bridge cycling, exposure to O2· had no effect
on force generation (24). These findings suggest that the
alteration produced by O2· exposure is not a
generalized or nonspecific effect on the contractile apparatus, leading
the authors to conclude that O2· does not promote
cross-bridge detachment or weaken the cross bridges but most likely
alters a part of the cross bridge that is not accessible in the
attached state, that is, either subsequent attachment, cross-bridge
kinetics, or ATPase activity (24). Recent studies by
Perkins et al. (32) have shown that specific modification
of the SH-1 subunit of myosin alters actomyosin ATPase activity and
reduces Ca2+ sensitivity in skinned psoas fibers.
Additionally, specific sulfhydryl modifying agents have been used to
modify myosin (20, 29, 48), and it has been suggested that
cross-linking of the SH-1 and SH-2 of myosin can induce decrements in
Fmax without altering Ca2+ sensitivity
(48), a finding that could explain our results.
Specific cysteine residues on actin have also been identified as potential sites of thiol modification by oxidants. However, studies show that, when actin is in its filamentous form, as in the skinned fiber preparation, the susceptibility of these residues to modification is reduced (23). Furthermore, although it has been suggested that troponin C is particularly sensitive to oxidative modification, Metzger and Moss (27) have shown that partial extraction of troponin C in skinned skeletal muscle fibers produces changes in both Ca2+ sensitivity and cooperativity of the contractile proteins. The absence of changes in Ca2+ sensitivity of the contractile proteins in the present study suggests that this is an unlikely mechanism. However, it is possible that biochemical modification of troponin C with resultant changes in the tertiary structure of the protein, and perhaps alteration in the Ca2+ sensitivity of troponin C per se, could be an explanation by which free radical-induced changes could alter the force generation of the contractile proteins (34). Whether or not other conformational changes in the other components of the troponin complex (i.e., troponin I or troponin T) could account for these findings is presently unknown.
Although it would be attractive to account for the findings in the present study by a single mechanism, it seems more likely that multiple sites on the proteins that comprise the contractile machinery are susceptible to redox modification. In fact, it is entirely possible that our findings may be due to several alterations that produce opposing effects on some aspects of the functional characteristics of the contractile proteins, while producing other effects that are synergistic.
Implications in Pathophysiological States
We and others have demonstrated that production of free radical species in muscle is increased in pathophysiological conditions, such as ischemia-reperfusion and sepsis (39, 46). In fact, we have recently established that, after endotoxin administration, force generation by intact diaphragm muscles, as well as the absolute force generated by the contractile proteins in Triton-skinned diaphragm fibers, is markedly reduced (42). Specifically, Fmax is significantly reduced without changes in calcium sensitivity. This relationship was shown to be true for all fiber types in the diaphragm, as well as in the soleus and extensor digitorum longus, indicating that the muscle dysfunction produced in sepsis is widespread and, in part, due to a direct effect on the contractile proteins. The present findings provide a potential mechanism for these previous observations.In conclusion, we have demonstrated that the contractile proteins are
susceptible to modification by O2· and ·OH.
Furthermore, exposure to these specific oxygen-derived free radicals
leads to alterations in physiological function, specifically, decreases
in the absolute force-generating capacity of the muscle without
alterations in calcium sensitivity. It is, therefore, possible that one
or more of these free radical species is responsible for producing
specific alterations in the contractile proteins, which leads to muscle
dysfunction in a variety of pathophysiological conditions. Albeit
potentially complex, we believe that further investigation is warranted
to elucidate the specific protein alteration or alterations
and the mechanisms by which these changes produce derangements in the
contractile apparatus.
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FOOTNOTES |
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Address for reprint requests and other correspondence: L. A. Callahan, Division of Pulmonary and Critical Care Medicine, MetroHealth Medical Center, Rm. H323, 2500 MetroHealth Drive, Cleveland, Ohio 44109 (E-mail: leighann_callahan{at}urmc.rochester.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 16 November 1999; accepted in final form 1 August 2000.
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TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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) in pCa 8.5 (n = 8).
, Baseline force vs. pCa relationship. A:
force is normalized to %maximum force under baseline conditions.
Similar changes in the force vs. pCa relationship were also seen with
exposure to 1,000 µM H2O2 [i.e., maximum
calcium-activated force (Fmax) was 94.4 ± 1.2% of
initial baseline force; n = 8] and in the in-time
control fibers (Fmax was 90.3 ± 1.7% of initial
baseline force; n = 8), indicating that exposure to
H2O2 does not significantly alter
Fmax. B: force is normalized to %maximum force
obtained at baseline and after H2O2 exposure to
demonstrate any alterations in the calcium concentration required for
half-maximal activation (Ca50). As shown,
H2O2 exposure has no significant effect on
Ca2+ sensitivity of the contractile proteins. Data points
are means ± SE. Data for 1,000 µM H2O2
and time-matched control fibers are reported in the text.
Significant difference compared with fibers treated with any
component of the generating system, with in-time control fibers, and
with the appropriate control conditions (X + XO + SOD),
P < 0.05.
