Superoxide Scavengers Improve Rat Pharyngeal Dilator

Muscle Performance
J. Richard Skelly1, Aidan Bradford2, James F. X. Jones1, and Ken D. OHalloran1
1
UCD School of Medicine and Medical Science, University College Dublin, Dublin, Ireland; and 2Department of Physiology and Medical Physics,
Royal College of Surgeons in Ireland, Dublin, Ireland

Obstructive sleep apnea is a common disorder associated with upper

airway muscle dysfunction. Agents that improve respiratory muscle
performance may be useful as an adjunct therapy. The aim of this
study was to examine the effects of antioxidants on rat pharyngeal
dilator muscle performance. Adult male Wistar rats were killed
humanely and isometric contractile properties of isolated sternohyoid muscle strips were examined in physiological salt solution at
358C in vitro. Muscle strips were incubated in tissue baths under
hyperoxic (95%O2/5%CO2) or hypoxic (95%N2/5%CO2) conditions
in the absence (control) or presence of the antioxidants: Nacetylcysteine (10 mM), Tiron (10 mM), or Tempol (10 mM).
Forcefrequency relationship was determined in response to supramaximal stimulation (10100 Hz in increments of 1020 Hz, train
duration: 300 ms). Isometric force was also recorded during repetitive muscle stimulation (40 Hz, 300 ms every 2 s for 2 min). Under
hyperoxic conditions, Tiron and Tempol, but not N-acetylcysteine,
significantly increased sternohyoid muscle force and caused a leftshift in the forcefrequency relationship. In addition, Tempol had
a significant positive inotropic effect over the initial 90 seconds of
repeated muscle activation. Hypoxia caused a significant decrease in
sternohyoid muscle force. Under hypoxic conditions, Tempol-incubated muscles generated significantly higher forces compared
with control muscles and showed improved performance in the early
phase of the fatigue trial. This study illustrates that superoxide
scavengers increase upper airway muscle force and that this effect
persists under hypoxic conditions. We conclude that antioxidant
treatment may be beneficial as a therapy in obstructive sleep apnea.
Keywords: antioxidants; hypoxia; superoxide scavengers; obstructive
sleep apnea

The human upper airway is compliant and vulnerable to collapse

because of its multifunctional role in breathing, swallowing, and
speech. Reflex activation of the pharyngeal dilator muscles
protects airway patency on a breath-by-breath basis and may be
especially important in alleviating obstruction of the upper
airway following airway collapse. Recurrent collapse of the upper
airway during sleep is a feature of obstructive sleep apnea (OSA),
a prevalent (1) and often debilitating respiratory disorder associated with significant cardiovascular and neurocognitive morbidities (2). The causes of upper airway obstruction during sleep
are multifactorial, but it is generally thought to occur as a result of
sleep-related decrements in upper airway muscle activity (3, 4) in
individuals with abnormal airway anatomy (2). Perhaps as many
as 20 skeletal muscles are involved in the control of airway caliber,

(Received in original form May 8, 2009 and in final form July 14, 2009)
This work was funded by the Health Research Board Ireland (RP/2006/140). J.R.S.
is a University College Dublin Ad Astra Research Scholar.
Correspondence and requests for reprints should be addressed to Ken OHalloran,
Ph.D., UCD School of Medicine and Medical Science, Room C228, Health Sciences
Centre, University College Dublin, Belfield, Dublin 4, Ireland. E-mail: ken.ohalloran@
ucd.ie
Am J Respir Cell Mol Biol Vol 42. pp 725731, 2010
Originally Published in Press as DOI: 10.1165/rcmb.2009-0160OC on July 27, 2009
Internet address: www.atsjournals.org

CLINICAL RELEVANCE
Obstructive sleep apnea (OSA) is caused by decrements in
upper airway dilator muscle tone in the transition from
wakefulness to sleep. Upper airway dilator muscle dysfunction is observed in patients with OSA and animal
models of the disease. It has been speculated that oxidative
stress leads to upper airway muscle damage in the patient
with OSA. This study illustrates that superoxide scavengers
increase upper airway muscle force and protect against
hypoxia-induced decreases in respiratory muscle performance. We conclude that antioxidant treatment may be
beneficial as an adjunct therapy in OSA.

and dysfunction of these accessory muscles of breathing may

contribute to airway narrowing, increased incidents of apnea, and
progression of OSA (5). Indeed, there is evidence of upper airway
muscle remodeling and dysfunction in OSA (6, 7) and in animal
models of the disorder (810). Pharmacotherapy for OSA is
considered a viable clinical option (11, 12), and naturally studies
have focused on agents that may increase airway caliber during
sleep by improving upper airway dilator muscle function (1316).
We hypothesized that antioxidants might prove beneficial in this
regard. Antioxidants have been shown to improve skeletal muscle
performance (17) and protect against hypoxia-induced muscle
impairment (18). To date, however, there have been no studies
examining the effects of antioxidants on upper airway muscle
contractile and endurance properties. We sought to determine
the effects of three antioxidant compounds on rat sternohyoid
muscle function in vitro under hyperoxic and hypoxic conditions.
We hypothesized that antioxidants would improve pharyngeal
dilator muscle performance especially under hypoxic conditions.

MATERIALS AND METHODS

In Vitro Muscle Preparation
Experiments were performed on 36 adult male Wistar rats (250370 g).
The animals were killed by a quick blow to the head followed by cervical
spinal cord transection. The paired sternohyoid muscles were separated
and placed in a bath at room temperature containing continuously
aerated (95% O2/5% CO2) physiological salt solution (PSS) containing
NaCl 120 mM, KCl 5 mM, Ca21 gluconate 2.5 mM, MgSO4 1.2 mM,
NaH2PO4 1.2 mM, NaHCO3 25 mM, glucose 11.5 mM, and d-tubocurarine
(25 mM). Longitudinal strips of muscle (12 mm in diameter) were placed
vertically in one of two water-jacketed organ baths at 358C aerated with
either 95% O2/5% CO2 for control studies or 95% N2/5% CO2 to create
tissue hypoxia (bath PO2 z 45 mm Hg). In preliminary studies, we
observed that sternohyoid force and endurance was optimum under
hyperoxic (95% O2) compared with normoxic conditions (21% O2); for
example, peak force was 9.7 6 0.8 N/cm2 (n 5 7) versus 17.7 6 2.1 N/cm2
(n 5 9), normoxia versus hyperoxia, respectively. Thus bath hyperoxia
provided the optimum control condition. The muscle strips were
positioned between a pair of platinum plate electrodes, with the base
fixed to an immobile hook and the other end tied to an isometric force

726

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010

transducer with nonelastic string. The position of the force transducer

could be adjusted by a micropositioner, thus altering the length of the
muscle strips. For each rat, two muscle strips were prepared: one for
hyperoxia and the other for hypoxia studied in parallel. Muscle function
was then assessed in one of four experimental groups (see below). Strips
were equilibrated in gassed PSS, with or without antioxidant for 5
minutes before initiating the experimental protocol.

Protocol
The optimum length was determined by adjusting the micropositioner
between intermittent stimulations. The muscle was then allowed a 5minute equilibration period. The single isometric twitch force, contraction time, half-relaxation time, forcefrequency relationship, and
performance during repeated stimulation were then determined in
response to electrical field stimulation. First, a single twitch was elicited
(supramaximal current, 1 ms duration). Twitch force, contraction time
(time to peak force), and half-relaxation time (time for peak force to
decay by 50%) were determined. Next, forcefrequency relationship
was determined by sequentially stimulating the muscle strips at 10, 20,
30, 40, 60, 80, and 100 Hz for 300 milliseconds at each stimulus
frequency, allowing a 2-minute recovery interval between each stimulus. Five minutes after this forcefrequency protocol, repeated muscle
contraction was induced by stimulation at 40 Hz with 300-millisecond
trains every 2 seconds for a period of 2 minutes. Studies of contractile
properties of sternohyoid muscle strips in hyperoxia and hypoxia were
performed in the absence (control, n 5 9) or presence of three different
antioxidants: N-acetyl cysteine (NAC 10 mM, n 5 9), 4,5-Dihydroxy1,3-benzenedisulfonic acid disodium salt (Tiron 10 mM, n 5 9), or 4hydroxy-2,2,6,6-tetramethylpiperidine-1-oxyl (Tempol 10 mM, n 5 9).
All drugs were purchased from Sigma-Aldrich Co., Dublin, Ireland and
prepared fresh each day.

to repeated activation we performed three sets of analyses. First, force

potentiation in the early phase of each trial was calculated as follows:
[(peak tetanic contraction/initial tetanic contraction 3 100) 2 100].
Second, each tetanic contraction was measured and specific force was
averaged in 10-second bins (i.e., five tetanic contractions) over the
initial 1 minute and in 30-second bins over the second minute. Third,
a performance index was calculated for the whole trial according to the
following: [average twitch amplitude of all 60 contractions/initial twitch
amplitude 3 100] modified from the work of Healy and colleagues (19).
Values are expressed as mean 6 SEM. Statistical comparisons between
groups were performed using one-way or two-way ANOVA (drug and
gas treatment) and Newman Keuls post hoc test as appropriate. P ,
0.05 was taken as significant in all tests.

RESULTS
Isometric Twitch and Peak Tetanic Force

Under hyperoxic conditions, Tempol caused a significant increase

in isometric twitch force compared with control data (Figures 1A
and 1C). A small positive inotropic effect on twitch force was
observed in hypoxia, for muscles incubated with Tiron (116%)
and Tempol (133%), but these increases failed to reach statistical
significance (Figures 1B and 1D). Antioxidant treatment had no
significant effect on contractile kinetics in either hyperoxia or
hypoxia. Peak tetanic force was significantly increased by Tempol
(Figures 2A and 2C) and Tiron, but not NAC, in hyperoxia, while
under hypoxic conditions, only Tempol was associated with
a significant increase in peak force (Figures 2B and 2D). Gas
Tempol interaction was deemed statistically insignificant with
P 5 0.0856 (two-way ANOVA).

Data Analysis
Specific force was calculated in N/cm2 of muscle cross-sectional area.
The contraction time and half-relaxation time were measured as
indices of isometric twitch kinetics. For the forcefrequency relationship, data were plotted as absolute stress (N/cm2) across the range of
stimulus frequencies employed in the study. In addition, nonlinear
regression (curve fit) analysis was employed (Graph Pad Prism) for
control and antioxidant groups, allowing us to determine minimum,
maximum, slope, and EF50 values (i.e., stimulus frequency producing
50% of peak force) for sternohyoid muscle under both hyperoxic and
hypoxic conditions. Finally, to assess muscle performance in response

ForceFrequency Relationship

In hyperoxia, both Tiron and Tempol caused significant increases

in force at high stimulation frequencies (Figures 3A3C). Furthermore, Tiron and Tempol significantly decreased the EF50
(Figure 3D) in hyperoxia, but only Tempol caused a significant
reduction of the EF50 in hypoxia (Figure 3H), in comparison to
control muscles. A similar positive inotropic effect was observed
in hypoxia (Figures 3E23G). NAC had no effect on any of the
measured parameters in hyperoxia or hypoxia (Figure 3).

Figure 1. (A) Original traces of isometric single twitch in hyperoxia in physiological salt solution only (PSS, Control)
and in the presence of 10 mM Tempol.
(B) Original traces of isometric single
twitch in hypoxia in control PSS and in
the presence of 10 mM Tempol. (C)
Values (mean 6 SEM) for isometric
single twitch force of sternohyoid muscle in control PSS (n 5 9) or PSS
containing an antioxidant (10 mM
Tempol [n 5 9], 10 mM Tiron [n 5 8],
or 10 mM NAC [n 5 9]) in hyperoxia.
Data show that Tempol caused a significant increase in force production relative to controls (2.3 6 0.4 versus *5.2 6
0.3 N/cm2, control [n 5 9] versus
Tempol [n 5 9], *P , 0.01, one-way
ANOVA). (D) Values (mean 6 SEM) for
isometric single twitch force of sternohyoid muscle in PSS only (Control, n 5
7) or PSS containing an antioxidant
(10 mM Tempol [n 5 8], 10 mM Tiron
[n 5 8], or 10 mM NAC [n 5 8]) in hypoxia. Data show that Tempol increased force production relative to control values, but this failed to reach
statistical significance.

Skelly, Bradford, Jones, et al.: Tempol Improves Upper Airway Muscle Performance

727

Figure 2. (A) Original traces of peak

force in hyperoxia in physiological salt
solution only (PSS, Control) and PSS in
the presence of 10 mM Tempol (traces
are superimposed for comparison). (B)
Original traces of peak force under hypoxic conditions. (C) Values (mean 6 SEM)
for peak tetanic tension of sternohyoid
muscle in PSS only (Control) or PSS
containing 10 mM Tempol. Data show
that Tempol increased peak force in
hyperoxia (16.5 6 1.9 versus 26.8 6
1.1 N/cm2, control [n 5 9] versus Tempol
[n 5 9], P , 0.001 one-way ANOVA).
The same is true in hypoxia (10.3 6 1.4
versus *15.1 6 1.7 N/cm2, hypoxia [n 5
7] versus hypoxia 1 Tempol [n 5 8], *P ,
0.05, one-way ANOVA, as shown in D).
Hypoxia caused a significant decrease in
force production (*P , 0.05, two-way
ANOVA) compared with control.

Performance during Repeated Stimulation

Data illustrating sternohyoid muscle performance during repeated stimulation in the absence (control) and presence of
antioxidants are shown for hyperoxia and hypoxia (Figure 4). In
hyperoxia, Tempol caused a significant increase in isometric force
production over the first 90 seconds of the trial (Figure 4C). Tiron
increased specific force during repeated stimulation, but this
failed to reach statistical significance (Figure 4). There was no
inotropic effect of NAC during repeated muscle activation
(Figure 4C). Force potentiation in response to repeated stimulation was observed in all sternohyoid muscle preparations, and this
phenomenon was enhanced in both Tiron- and Tempol-treated
muscle strips compared with control (Figure 5A). The performance index of the muscles was significantly increased by Tiron
but not by NAC or Tempol in hyperoxic conditions (Figure 5C).
Under hypoxic conditions, Tempol significantly increased sternohyoid muscle force over the initial 50 seconds of fatigue (Figure
4). As before, Tiron was associated with a positive inotropic effect
that failed to reach statistical significance, and NAC had no
discernible effect on muscle performance (Figure 4F). The
performance index of the muscles was significantly increased by
Tiron alone in hypoxic conditions (Figure 5D).

DISCUSSION
OSA afflicts approximately 4% of the adult male population (1),
with greater prevalence in the elderly and obese, and it is
associated with significant cardiovascular and neurocognitive
morbidities. There has been considerable interest over the years
in the neuromuscular mechanisms that regulate pharyngeal
airway caliber, and it is now widely accepted that the striated
muscles of the upper airway are critical for the control and
maintenance of pharyngeal airway patency. Several research
groups have focused their attention on the contractile and
endurance properties of the upper airway muscles, since the
mechanical performance of these muscles is a critical determinant
of pharyngeal airway stability. The structural and functional
properties of the pharyngeal dilator muscles are well described.
These muscles have a high proportion of fast twitch fibers (20, 21),

which have relatively poor fatigue resistance (14, 2224). As

fatigue of the upper airway muscles is implicated in OSA, it has
been suggested that upper airway occlusion during sleep in
patients with OSA may result from impaired muscle function in
addition to state-dependent reductions in cranial motor drive to
the upper airway musculature. As such, improved muscle performance during sleep in patients with OSA may help reduce the
incidence and/or severity of occlusive events.
Pharmacological therapy has been suggested as a clinical
strategy in the treatment of OSA (11, 12) and agents that improve
upper airway muscle performance could serve as viable adjunct
therapies in patients with sleep-disordered breathing. Previous
investigators have studied the action of potassium channel
blockers (15, 25), theophylline (13), almitrine (14), and nicotine
(16) on isolated muscle function with mixed results. Of note,
almitrine was shown to improve geniohyoid and sternohyoid
muscle endurance (14) and potassium channel blockers increased
sternohyoid muscle force. Since reactive oxygen species (ROS)
are implicated in skeletal muscle function (15, 26) and antioxidants have been shown to improve muscle performance (17, 27)
we sought to examine the effects of antioxidants on sternohyoid
muscle function.
We chose to study the sternohyoid muscle, as it is one of
a group of phasic inspiratory muscles that influence the position of
the hyoid bone. Simultaneous contraction of infrahyoid and
suprahyoid muscles dilates the airway. Furthermore, an inverse
relationship between upper airway volume and hyoid muscle
length has been shown (28, 29). The muscle is readily accessible
and has fibers running in a uniform direction, making it suitable
for in vitro study. Furthermore, sternohyoid muscle dysfunction
has been shown in animal models of sleep-disordered breathing
(10, 30). The main finding of the present study is that the
superoxide scavenger Tempol, and to a lesser extent Tiron, have
positive inotropic effects on rat sternohyoid muscle under both
hyperoxic and hypoxic conditions. Conversely, the thiol donor
NAC had no significant effect on pharyngeal dilator muscle
performance. NAC is a well-known nonspecific antioxidant with
proven actions in humans and animal models. NAC scavenges
hydroxyl radicals and may increase the activity of other endog-

728

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010

Figure 3. Original traces of force

frequency relationship in hyperoxia in
physiological salt solution only (PSS,
Control) (A) and PSS containing
10 mM Tempol (B). The (-//-) sign indicates 2-minute intervals in the recordings. (C) Group values (mean 6 SEM) for
force frequency under hyperoxic conditions. Both Tiron (22.7 6 1.7 N/cm2,
n 5 8) and Tempol (26.8 6 1.1 N/cm2,
n 5 9) caused significant increases
in sternohyoid muscle force at higher
frequencies in comparison to control
(16.5 6 1.9 N/cm2, n 5 9; P , 0.001,
one-way ANOVA). D shows that Tempol
caused a significant reduction in EF50
compared with control in hyperoxia
(*48.5 6 1.6 Hz and 66.3 6 3.6 Hz for
Tempol [n 5 9] and Control [n 5 9],
respectively, one-way ANOVA; (*P ,
0.01). Original traces of forcefrequency
relationship in hypoxia in physiological
salt solution only (PSS, Control) (E) and
PSS containing 10 mM Tempol (F). (G)
Group values (mean 6 SEM) for force
frequency under hypoxic conditions.
Tempol caused significant increases in
sternohyoid muscle force at higher frequencies in comparison to control (*P ,
0.01, one-way ANOVA). H shows that
Tempol (n 5 8) caused a significant reduction in EF50 compared with control
(n 5 7) in hypoxia (49.1 6 2.2 Hz and
76.8 6 0.8 Hz, respectively (P , 0.001,
one-way ANOVA).

enous intracellular antioxidant enzymes such as superoxide

dismutase (SOD). Also, since NAC supports glutathione biosynthesis by functioning as a cysteine donor, NAC has indirect
antioxidant effects. NAC has been shown to be effective in
inhibiting skeletal muscle fatigue (31, 32). Furthermore, NAC
protects diaphragm muscle function during hypoxia (33). Thus,
the lack of effect of NAC in this study was unexpected. Interestingly, NAC (0.110 mM) was also ineffective in studies of
sternohyoid muscle under normoxic conditions (data not shown).
However, thiol donors are not always effective in improving
muscle performance. For example, conflicting with earlier reports, Wright and colleagues (18) and Healy and coworkers (19)
reported no effect of NAC (< 18 mM) on skeletal muscle
function, and concentrations in excess of 30 mM depress muscle
force (34, 35). The present study clearly demonstrates that NAC
has no effect on pharyngeal dilator muscle function. While one

interpretation of our results might be that NAC, which is

approved for use in humans, has limited functional use in
protecting upper airway muscles during hypoxia, it should be
noted that chronic administration of NAC was shown to be
protective in a rat model of chronic intermittent hypoxia-induced
respiratory muscle impairment (10), consistent with other models
of free radicalmediated muscle dysfunction (26, 36). It is
conceivable that chronic administration of NAC improves
muscle performance as a result of increased glutathione synthesis, whereas acute in vitro administration of NAC, as in the
present study, may not have increased endogenous antioxidant
defenses.
Unlike NAC, the superoxide scavengers Tiron and Tempol
improved sternohyoid muscle force and performance. Tiron has
been shown to improve rat diaphragm endurance and protect rat
diaphragm muscle contractile function during hypoxia (18). In

Skelly, Bradford, Jones, et al.: Tempol Improves Upper Airway Muscle Performance

729

Figure 4.
Original
traces of repeated muscle stimulation in hyperoxia in physiological salt
solution only (PSS, Control) (A) and PSS in the
presence of 10 mM
Tempol (B). C illustrates
group data (mean 6
SEM) showing that Tempol (n 5 9) significantly
increases sternohyoid
muscle force production
throughout the first 90
seconds of the trial compared with control (n 5
9, #P , 0.05, P , 0.001
one-way ANOVA). Original traces of repeated
muscle stimulation in
hypoxia in physiological
salt solution only (PSS,
Control) (D) and PSS in
the presence of 10 mM
Tempol (E ). F illustrates
group data (mean 6
SEM) showing that
Tempol (n 5 8) increases
sternohyoid
muscle force production
throughout the first 50
seconds of fatigue compared with control (n 5
7, #P , 0.05, P , 0.001,
one-way ANOVA).

intact isolated rat and mouse extensor digitorum longus muscle

preparations, Tempol was shown to increase the maximum Ca21activated force and ameliorate decreases in tetanic force related
to heat stress (37). In addition, Tempol maintains force in
repetitively stimulated rat extensor digitorum longus muscle at
378C (37, 38). We observed a positive inotropic effect of Tempol
and Tiron, which was maintained during repeated muscle activation. Tempol also proved to be effective during hypoxia, consistent with observations that superoxide scavengers protect
diaphragm muscle contractile function in severe hypoxia (18,
33). It is important to note, however, that there may be complex
doseresponse relationships for the agents tested in this study. As
such, direct comparisons of the actions of a single concentration
of Tiron or Tempol on muscle function may not be appropriate. It
would be useful to determine the effects of these agents on upper
airway muscle function across a broad range of concentrations.
While the effects described in this study are likely to be beneficial
in terms of the maintenance and/or restoration of airway caliber,
the mechanism of action of the antioxidants is unclear. ROS are
formed in the mitochondria of skeletal muscle in response to
muscle activation, heat stress, and ischemia-reperfusion injury
(39, 40), and free radicalmediated muscle dysfunction is well
described (26). The positive inotropic effect of Tempol presumably relates to increases in myoplasmic calcium concentration and/or increased sensitivity of the myofibrillar contractile
proteins to calcium. The sarcoplasmic reticulum calcium release
and re-uptake channels are redox sensitive (36, 41), and ROS may
also affect cross-bridge cycling (41, 42). Of note, contractile
kinetics were unaffected by superoxide scavengers in this study
under all conditions, whereas isometric force was significantly

elevated. This suggests that the facilitatory effect of the drug is

largely dependent on improved sensitivity of the myofibrillar
proteins to calcium.
Opinion is divided on whether ROS are generated in greater
amounts during hypoxia. It is generally taken that in low O2
environments it is difficult to form the ROS precursorsuperoxide. Increased ROS generation seems more likely during
hyperoxia or the reoxygenation that occurs during intermittent
hypoxia. There are reports, however, that, somewhat paradoxically, ROS production is elevated during hypoxia (43, 44) and
may contribute to reduced muscle performance under hypoxic
conditions. In our study, muscle force recorded in hypoxia was
significantly increased by Tempol, resulting in values similar to
those recorded under control conditions. It is tempting, but
perhaps too simplistic an interpretation, to suggest that hypoxia-induced oxidative stress was abrogated by antioxidant
treatment. In hyperoxia, the inotropic effect of Tempol was
comparable to that seen in hypoxia (i.e., there was no druggas
interaction). This suggests that ROS turnover and associated
oxidative stress was similar in the two conditions and may
therefore be less related to the bath O2 tension and perhaps more
dependent on other factors such as temperature or muscle
activation, both of which can increase ROS production (40, 45,
46). A limitation of the present study is that superoxide production in sternohyoid muscle was not measured. Previous work
(45, 46) has shown that there is basal superoxide production in
passive (resting) muscle, which increases with muscle contraction
and is maximum in response to fatiguing contractions reaching
values of 0.7 nmol/minute (45). Exogenous SOD was shown to
prevent this increased production (45). Measurements of tissue

730

AMERICAN JOURNAL OF RESPIRATORY CELL AND MOLECULAR BIOLOGY VOL 42 2010

Figure 5. Group data (mean 6 SEM) for

force potentiation during repeated muscle stimulation trials in both hyperoxic
(A) and hypoxic (B) conditions. In hyperoxia (A), Tempol (n 5 9) and Tiron (n 5
8) increased force potentiation in comparison to control (n 5 9, #77.4 6 8.9%,
#76.5 6 11.0% versus 48.8 6 3.2%,
respectively; #P , 0.05, one-way
ANOVA). Hypoxia decreased potentiation from control values (1P , 0.0001,
two-way ANOVA). C and D show group
data for performance index throughout
the repeated stimulation protocol in
both hyperoxic (C) and hypoxic (D)
conditions. Performance index was enhanced by Tiron (n 5 8) in hyperoxia
compared with control (n 5 9) (C,
#133.2 6 7.3% versus 109.5 6 2.0%,
respectively, #P , 0.05, one-way
ANOVA). In hypoxia, Tiron (n 5 8) increased performance index compared
with control (n 5 7) (#100.1% 6 3.9
versus 80.3% 6 3.8 respectively, #P ,
0.05, one-way ANOVA). Hypoxia
inhibited performance index causing significant reductions from control values
(1P , 0.0001, two-way ANOVA).

superoxide under control and hypoxic conditions in the absence

and presence of antioxidants would have been useful.
Whatever the mechanism of action, the positive inotropic
effect of the superoxide scavengers suggests that antioxidant
therapy may be beneficial in the treatment of respiratory disorders associated with muscle dysfunction. Encouragingly, the
superoxide scavengers caused a left-shift in the forcefrequency
relationship, as illustrated by the significant decreases in the EF50
values. This suggests that in the presence of Tempol, lower motor
neurone firing rates would be necessary to achieve muscle
activation in vivo, which is especially relevant to OSA, as
decrements in upper airway motor drive predispose to collapse
of the compliant pharyngeal airway during sleep. Thus agents
such as Tempol may help improve control of pharyngeal airway
caliber during sleep by virtue of both an increase in upper airway
muscle sensitivity to a given motor outflowpresumably helping
to stabilize the airwayand increased force of contraction in
response to high stimulus rates, which may be especially relevant
to re-opening the airway following occlusion. Interestingly, instantaneous motor unit firing rates in excess of 100 Hz have been
recorded in human genioglossus (47) and sternohyoid (48)
muscles during sustained contractions; thus the positive inotropic
effect of Tempol at high stimulus frequencies in this study is
physiologically relevant assuming a similar action of the drug in
humans. While one could argue that antioxidants (or any
inotropic agents) that are ineffective across the lower range of
stimulus frequencies (as in this study) are unlikely to prevent
occlusions during sleep-induced upper airway muscle hypotonia,
it is plausible that such agents would expedite airway re-opening
following collapse, thereby limiting the severity and duration of
obstructive events. In this way, such agents might help reduce the
apnea-hypopnoea index in patients with OSA. We concede,
however, that one should exercise caution in extrapolating this
data to the human patient. Furthermore, it is important to
acknowledge that fatigue was accelerated by Tempol, and the

positive inotropic effect of the drug was lost in this study after 90
seconds of repeated muscle activation. This finding suggests that
there may be significant limitations to the efficacy of antioxidant
treatment in human patients with respiratory muscle disorders. If
force decline with repeated stimulation of the upper airway
muscles is largely unaffected by superoxide scavengers in the
long term, then one could argue that antioxidants may have little
therapeutic value. It would be interesting to explore whether
these agents are effective in improving recovery from fatigue,
which would be clinically relevant. Also, there would be great
value in assessing the effects of superoxide scavengers on upper
airway muscle function in an in vivo model. On balance, however,
we speculate that antioxidant therapy would prove beneficial in
the treatment of OSA. Hence, studies of the efficacy of antioxidants in patients with sleep-disordered breathing are clearly
warranted. As an added benefit, antioxidant supplementation is
likely to limit oxidative stress associated with recurrent hypoxemiaa hallmark feature of OSA.
To summarize, we examined the action of antioxidant compounds on pharyngeal dilator muscle performance. Of the three
antioxidants tested, Tempol, a superoxide scavenger, was shown
to be most effective resulting in positive inotropy and increased
sensitivity to electrical stimulation, as shown by the left-shift in
forcefrequency relationship (i.e., a decrease in EF50 value).
Tempol also improved pharyngeal dilator muscle function during
hypoxia. Such changes, if they could be reproduced in humans,
would presumably be of benefit in the treatment of OSA, where
state-dependent decreases in neural drive to the upper airway
muscles leads to airway narrowing and occlusion (49). In addition,
OSA is now widely recognized as an oxidative stress disorder (50,
51) and antioxidant therapies have been shown to protect against
the adverse effects of intermittent hypoxia in animal models of
human sleep apnea (10, 52). Together, the results of these studies
highlight the potential therapeutic value of antioxidants in the
treatment of sleep-disordered breathing.

Skelly, Bradford, Jones, et al.: Tempol Improves Upper Airway Muscle Performance
Conflict of Interest Statement: None of the authors has a financial relationship
with a commercial entity that has an interest in the subject of this manuscript.

References
1. Young T, Palta M, Dempsey J, Skatrud J, Weber S, Badr S. The
occurrence of sleep-disordered breathing among middle-aged adults.
N Engl J Med 1993;328:12301235.
2. White DP. Pathogenesis of obstructive and central sleep apnea. Am J
Respir Crit Care Med 2005;172:13631370.
3. Wheatley MW Jr, Tangel DJ, White DP. Influence of sleep on
genioglossus muscle activation by negative pressure in normal men.
Am Rev Respir Dis 1993;148:597605.
4. Horner RL, Innes JA, Morrell MJ, Shea SA, Guz A. The effect of sleep
on reflex genioglossus muscle activation by stimuli of negative airway
pressure in humans. J Physiol 1994;476:141151.
5. Boyd JH, Petrof BJ, Hamid Q, Fraser R, Kimoff RJ. Upper airway
muscle inflammation and denervation changes in obstructive sleep
apnea. Am J Respir Crit Care Med 2004;170:541546.
6. Stauffer JL, Buick MK, Bixler EO, Sharkey FE, Abt AB, Manders
EK, Kales A, Cadieux RJ, Barry JD, Zwillich CW. Morphology of
the uvula in obstructive sleep apnea. Am Rev Respir Dis 1989;140:
724728.
7. Serie`s F, Simoneau S, St Pierre S, Marc I. Characteristics of the
genioglossus and musculus uvulae in sleep apnea hypopnea syndrome and in snorers. Am J Respir Crit Care Med 1996;153:1870
1874.
8. Bradford A, McGuire M, OHalloran KD. Does episodic hypoxia affect
upper airway dilator muscle function? Implications for the pathophysiology of obstructive sleep apnoea. Respir Physiol Neurobiol 2005;147:
223234.
9. Carrera M, Barbe F, Sauleda J, Tomas M, Gomez C, Agusti AG.
Patients with obstructive sleep apnea exhibit genioglossus dysfunction
that is normalized after treatment with continuous positive airway
pressure. Am J Respir Crit Care Med 1999;159:19601966.
10. Dunleavy M, Bradford A, OHalloran K. Oxidative stress impairs upper
airway muscle endurance in an animal model of sleep-disordered
breathing. Adv Exp Med Biol 2008;605:458462.
11. Hudgel DW, Thanakitcharu S. Pharmacologic treatment of sleepdisordered breathing. Am J Respir Crit Care Med 1998;158:691699.
12. Veasey SC. Serotonin agonists and antagonists in obstructive sleep
apnea: therapeutic potential. Am J Respir Med 2003;2:2129.
13. Van Lunteren E, Vafaie H, Miller MJ. Effects of theophylline on
pharyngeal dilator and diaphragm muscle contractile properties.
Respiration 1996;63:8893.
14. Cantillon D, Bradford A. Effect of almitrine on upper airway muscle
contraction in young and old rats. Eur J Pharmacol 2001;412:187194.
15. Van Lunteren E, Vafaie H, Moyer M. Changes in pharyngeal respiratory
muscle force produced by K1 channel blockade. Respir Physiol 1995;
99:331340.
16. OHalloran KD. Effects of nicotine on rat sternohyoid muscle contractile properties. Respir Physiol Neurobiol 2006;150:200210.
17. Reid MB. Free radicals and muscle fatigue: of ros, canaries, and the ioc.
Free Radic Biol Med 2008;44:169179.
18. Wright VP, Klawitter PF, Iscru DF, Merola AJ, Clanton TL. Superoxide
scavengers augment contractile but not energetic responses to hypoxia in rat diaphragm. J Appl Physiol 2005;98:17531760.
19. Healy CF, McMorrow C, OHerlihy C, OConnell PR, Jones JF.
External anal sphincter fatigue is not improved by N-acetylcysteine
in an animal model. Neurogastroenterol Motil 2008;20:719724.
20. Malhotra A, White DP. Obstructive sleep apnoea. Lancet 2002;360:237245.
21. Dick TE, van Lunteren E. Fiber subtype distribution of pharyngeal
dilator muscles and diaphragm in the cat. J Appl Physiol 1990;68:
22372240.
22. Cantillon D, Bradford A. Effects of age and gender on rat upper airway
muscle contractile properties. J Gerontol A Biol Sci Med Sci 2000;55:
B396B400.
23. McGuire M, MacDermott M, Bradford A. Effects of chronic episodic
hypoxia on rat upper airway muscle contractile properties and fibertype distribution. Chest 2002;122:10121017.
24. El-Khoury R, OHalloran KD, Bradford A. Effects of chronic hypobaric
hypoxia on contractile properties of rat sternohyoid and diaphragm
muscles. Clin Exp Pharmacol Physiol 2003;30:551554.
25. Van Lunteren E, Vafaie H. Force potentiation in respiratory muscles:
comparison of diaphragm and sternohyoid. Am J Physiol 1993;264:
R1095R1100.

731

26. Supinski G. Free radical induced respiratory muscle dysfunction. Mol

Cell Biochem 1998;179:99110.
27. Reid MB, Haack KE, Franchek KM, Valberg PA, Kobzik L, West MS.
Reactive oxygen in skeletal muscle: I. Intracellular oxidant kinetics
and fatigue in vitro. J Appl Physiol 1992;73:17971804.
28. Van de Graaff WB, Gottfried SB, Mitra J, van Lunteren E, Cherniack
NS, Strohl KP. Respiratory function of hyoid muscles and hyoid arch.
J Appl Physiol 1984;57:197204.
29. Van Lunteren E, Haxhiu MA, Cherniack NS. Relation between upper airway volume and hyoid muscle length. J Appl Physiol 1987;63:14431449.
30. OHalloran KD, McGuire M, OHare T, Bradford A. Chronic intermittent asphyxia impairs rat upper airway muscle responses to acute
hypoxia and asphyxia. Chest 2002;122:269275.
31. Ferreira LF, Reid MB. Muscle-derived ros and thiol regulation in muscle
fatigue. J Appl Physiol 2008;104:853860.
32. Shindoh C, DiMarco A, Thomas A, Manubay P, Supinski G. Effect of Nacetylcysteine on diaphragm fatigue. J Appl Physiol 1990;68:21072113.
33. Mohanraj P, Merola AJ, Wright VP, Clanton TL. Antioxidants protect
rat diaphragmatic muscle function under hypoxic conditions. J Appl
Physiol 1998;84:19601966.
34. Diaz PT, Brownstein E, Clanton TL. Effects of N-acetylcysteine on
in vitro diaphragm function are temperature dependent. J Appl
Physiol 1994;77:24342439.
35. Khawli FA, Reid MB. N-acetylcysteine depresses contractile function and
inhibits fatigue of diaphragm in vitro. J Appl Physiol 1994;77:317324.
36. Supinski GS, Callahan LA. Free radical-mediated skeletal muscle dysfunction in inflammatory conditions. J Appl Physiol 2007;102:20562063.
37. Edwards JN, Macdonald WA, van der Poel C, Stephenson DG. O2(*-)
production at 37 degrees C plays a critical role in depressing tetanic
force of isolated rat and mouse skeletal muscle. Am J Physiol Cell
Physiol 2007;293:C650C660.
38. Van der Poel C, Edwards JN, Macdonald WA, Stephenson DG.
Mitochondrial superoxide production in skeletal muscle fibers of
the rat and decreased fiber excitability. Am J Physiol Cell Physiol
2007;292:C1353C1360.
39. Pattwell D, McArdle A, Griffiths RD, Jackson MJ. Measurement of free
radical production by in vivo microdialysis during ischemia/reperfusion injury to skeletal muscle. Free Radic Biol Med 2001;30:979985.
40. Zuo L, Christofi FL, Wright VP, Liu CY, Merola AJ, Berliner LJ,
Clanton TL. Intra- and extracellular measurement of reactive oxygen
species produced during heat stress in diaphragm muscle. Am J
Physiol Cell Physiol 2000;279:C1058C1066.
41. Andrade FH, Reid MB, Westerblad H. Contractile response of skeletal
muscle to low peroxide concentrations: myofibrillar calcium sensitivity as a likely target for redox-modulation. FASEB J 2001;15:309311.
42. Hertelendi Z, Toth A, Borbely A, Galajda Z, van der Velden J, Stienen
GJ, Edes I, Papp Z. Oxidation of myofilament protein sulfhydryl
groups reduces the contractile force and its Ca21 sensitivity in human
cardiomyocytes. Antioxid Redox Signal 2008;10:11751184.
43. Vanden Hoek TL, Li C, Shao Z, Schumacker PT, Becker LB. Significant
levels of oxidants are generated by isolated cardiomyocytes during
ischemia prior to reperfusion. J Mol Cell Cardiol 1997;29:25712583.
44. Prabhakar NR, Kumar GK, Nanduri J, Semenza GL. Ros signaling in
systemic and cellular responses to chronic intermittent hypoxia.
Antioxid Redox Signal 2007;9:13971403.
45. Kolbeck RC, She Z-W, Callahan LA, Nosek TM. Increased superoxide
production during fatigue in the perfused rat diaphragm. Am J Respir
Crit Care Med 1997;156:140145.
46. Reid MB, Shoji T, Moody MR, Entman ML. Reactive oxygen in skeletal
muscle: II. Extracellular release of free radicals. J Appl Physiol 1992;
73:18051809.
47. Bailey EF, Rice AD, Fuglevand AJ. Firing patterns of human genioglossus motor units during voluntary tongue movement. J Neurophysiol 2007;97:933936.
48. Farina D, Falla D. Discharge rate of sternohyoid motor units activated
with surface emg feedback. J Neurophysiol 2009;101:624632.
49. Veasey SC. Molecular and physiologic basis of obstructive sleep apnea.
Clin Chest Med 2003;24:179193.
50. Lavie L. Obstructive sleep apnoea syndrome: an oxidative stress
disorder. Sleep Med Rev 2003;7:3551.
51. Prabhakar NR. Sleep apneas: an oxidative stress? Am J Respir Crit Care
Med 2002;165:859860.
52. Xu W, Chi L, Row BW, Xu R, Ke Y, Xu B, Luo C, Kheirandish L, Gozal
D, Liu R. Increased oxidative stress is associated with chronic intermittent hypoxia-mediated brain cortical neuronal cell apoptosis in
a mouse model of sleep apnea. Neuroscience 2004;126:313323.