© 2002 American Thoracic Society
Adaptation of Upper Airway Muscles to Chronic Endurance ExerciseDepartment of Exercise and Sport Sciences and Department of Physiology, Center for Exercise Science, University of Florida, Gainesville, Florida; and Department of Exercise and Nutrition Sciences, and Center for Sleep Disorders Research, University at Buffalo, Buffalo, New York Correspondence and requests for reprints should be addressed to Scott K. Powers, Ph.D., Department of Exercise and Sport Sciences, Room 33-FLG, Center for Exercise Science, University of Florida, Gainesville, FL 32611. E-mail: spowers{at}hhp.ufl.edu
We tested the hypothesis that chronic endurance exercise is associated with the recruitment of four major upper airway muscles (genioglossus, digastric, sternohyoid, and omohyoid) and results in an increased oxidative capacity and a fast-toward-slow shift in myosin heavy chain (MHC) isoforms of these muscles. Female Sprague–Dawley rats (n = 8; 60 days old) performed treadmill exercises for 12 weeks (4 days/week; 90 minutes/day). Age-matched sedentary female rats (n = 10) served as control animals. Training was associated with an increase (p < 0.05) in the activities of both citrate synthase and superoxide dismutase in the digastric and sternohyoid muscles, as well as in the costal diaphragm. Compared with the control animals, Type I MHC content increased (p < 0.05) and Type IIb MHC content decreased (p < 0.05) in the digastric, sternohyoid, and diaphragm muscles of exercised animals. Training did not alter (p > 0.05) MHC phenotype, oxidative capacity, or antioxidant enzyme activity in the omohyoid or genioglossus muscle. These data indicate that endurance exercise training is associated with a fast-to-slow shift in MHC phenotype together with an increase in both oxidative and antioxidant capacity in selected upper airway muscles. It seems possible that this exercise-mediated adaptation is related to the recruitment of these muscles as stabilizers of the upper airway.
Key Words: respiratory muscles • oxidative • antioxidant • myosin • upper airway muscles
Maintenance of pharyngeal patency during breathing requires the coordinated activity of upper airway and thoracic respiratory muscles (1, 2). During inspiration, subatmospheric pressures are produced in the upper airway as a result of inspiratory muscle contraction. The tendency for the pharyngeal lumen to collapse is opposed by the activation and contraction of the upper airway muscles including dilators, such as the sternohyoid (SH) and the omohyoid (OH), and pharyngeal lumen regulators, such as the geniglossus (GG) and digastric (DG) muscles (2).
Ventilatory muscles, particularly the diaphragm, work at higher work rates during exercise training (3). In response to this overload stimulus, metabolic adaptations occur in the diaphragm that improve fatigue resistance (4). These adaptations include improvements in muscle oxidative and antioxidant capacity (5, 6) and a shift in the myosin heavy chain (MHC) phenotype to that of a greater expression of slow isoforms (Type IIb On the basis of the knowledge that exercise increases ventilatory requirements, it seems plausible that endurance exercise will result in the recruitment of respiratory upper airway muscles such as the SH, OH, GG, and DG to increase upper airway diameter and reduce airway resistance. Hence, we hypothesize that exercise training will elevate the recruitment of these muscles and therefore will improve the oxidative capacity of these muscles and promote a shift in fast-toward-slow MHC isoforms similar to the exercise-induced adaptations that occur in the diaphragm. Further, we postulate that endurance training will promote an increase in the antioxidant capacity of upper airway muscles that will result in lower resting levels of lipid peroxidation in the trained muscles. The rationale for this hypothesis is as follows. As aerobic metabolism is increased in contracting locomotor or respiratory muscles during exercise, the production of radicals and other reactive oxygen species (ROS) is also increased (7). Further, high rates of ROS production during intense muscular activity can promote oxidative damage in muscles (e.g., lipid peroxidation) (8, 9). In an effort to reduce this type of oxidative injury, cells increase their antioxidant defenses in response to repeated exposure to oxidative stress (8). Indeed, regular endurance exercise training has been shown to elevate the antioxidant capacity of the diaphragm and locomotor muscles (10). It follows that chronic endurance exercise could enhance antioxidant enzyme activities in contracting respiratory upper airway muscles as an adaptive response to the oxidative stress associated with exercise. Therefore, to test the aforementioned hypotheses, these experiments were designed to determine whether (1) endurance training–induced increases in the oxidative capacity and MHC phenotype of the respiratory upper airway musculature (i.e., SH, OH, DG, and GG muscles) are similar to those of the diaphragm; and (2) endurance exercise training promotes an increase in the activity of primary antioxidant enzymes and reduces resting levels of lipid peroxidation in respiratory upper airway muscles.
These experiments followed the guidelines established by the American Physiological Society and were approved by the Animal Care and Use Committee of both the University of Florida and the University at Buffalo. Sprague–Dawley rats were randomly divided into three experimental groups: (1) control, sedentary (n = 10); (2) endurance exercise–trained (n = 10); and (3) acute exercise with measurement of electromyographic (EMG) activity of upper airway muscles (n =12). The animals assigned to the exercise-training group were exercised during a 12-week, progressive treadmill protocol. Animals exercised 4 days/week (90 minutes/day) at approximately 75% maximal oxygen con- sumption. The protocol began with animals running at 30 m/minute at 0% grade. Increasing the treadmill grade by 2.5% every 2 weeks increased the exercise work rate over time.
Biochemical Measurements Samples were homogenized and centrifuged, and supernatants were assayed for the oxidative enzyme citrate synthase (CS, E.C. 4.1.3.7) and antioxidant enzymes glutathione peroxidase (GPX, E.C. 1.11.1.9) and superoxide dismutase (SOD, E.C. 1.15.1.1). The activities of the manganese dependent isoform of SOD (Mn-SOD) and the copper–zinc dependent cytosolic isoform (CuZn-SOD) were also determined. CS, SOD, and GPX were measured using the methods described by Srere (11), Flohe and Gunzler (12), and Oyanagui (13). To determine if a training-induced upregulation of the antioxidant enzyme activities in upper airway muscles was associated with protection against ROS-mediated lipid damage, lipid hydroperoxide contents of all muscles were measured as described by Hermes-Lima and coworkers (14). Protein concentrations of all homogenates were determined using the Bradford technique (15). All enzyme activities and lipid peroxide concentrations were normalized to the protein concentration of the homogenate. Electrophoretic separation to determine MHC composition was achieved using the sodium dodecyl sulfate–polyacrylamide gel electrophoresis procedure described by Talmadge and Roy (16). The relative concentrations of MHC isoforms were determined by scanning the gels using a computerized image analysis system.
Measurement of Upper Airway EMG Activity during Exercise The EMG activity pattern of upper airway muscles was recorded 15 to 27 days postsurgery. Before testing, all animals were habituated to the treadmill on three to four occasions. On the day of recording, the animal was connected to three channels of an eight-channel polygraph (MT-950 chart recorder/Grass P511 preamplifiers; Astro-Med, West Warwick, RI). Raw EMG signals were band-pass filtered between 10 and 30 Hz. The animal was then placed on the treadmill and allowed to move freely within the treadmill chamber for 20–30 minutes. The gains on the amplifiers, as well as the electrode pairings used for recording, were set during this initial quiet period and, once set, remained unchanged for the duration of the study. Muscle recruitment during nonexercise was then recorded for 10 minutes. The treadmill was then turned on, and the speed was progressively increased to 19–22 m/minute over a 2-minute period. Muscle recruitment was recorded continuously during the run test for 10 minutes. At the end of 10 minutes, the treadmill was abruptly turned off, whereas EMG recording continued for 2–5 minutes postexercise. This transition period between hyperpnea during locomotion to hyperpnea without locomotion (immediate postexercise events) was used to distinguish respiratory from locomotory activation.
Statistical Analysis
Animal Body Weights Eight animals successfully completed the 12-week training program. Both sedentary and trained groups showed a similar increase in body weight during the 12-week experimental period, indicating that exercise training did not affect weight gain throughout the 12-week study. The initial and final body weights of the animals used in the training study were (mean ± SEM): (1) control animals— 257 ± 5 g (initial), 296 ± 5 g (final); and (2) trained animals— 259 ± 6 g (initial), 283 ± 7 g (final).
Antioxidant and Oxidative Enzyme Activities
Lipid peroxidation values for all airway muscles are given in Table 2 . The levels of lipid hydroperoxides were higher (p < 0.05) in the SH, DG, and DIA of the control group compared with their trained counterparts.
MHC Analysis MHC profiles for the costal diaphragm and the upper airway muscles are illustrated in Figure 1 . Note that training was associated with a reduction (p < 0.05) in Type IIb MHC content in the SH and DIA muscles with a corresponding increase in the content of Types IIa and I MHC. Similarly, the content of Type IIb MHC was reduced in the DG of trained animals with a corresponding increase in Type IId/x and I MHC isoforms. No significant differences existed between experimental groups (p > 0.05) in the MHC content in either the GG or the OH muscle.
EMG Activity during Exercise EMG studies were conducted on 12 male Sprague–Dawley rats weighing 362 ± 5.3 g. Our EMG measurements were not designed to provide quantitative data on upper airway muscle recruitment during exercise but rather were designed to provide qualitative insight into the pattern of upper airway muscle recruitment during treadmill running. Figure 2 illustrates the EMG activity at rest and during exercise in two experimental animals. During the pre-exercise period, the PS was phasically active in 10 of 12 rats, whereas it was tonically active in the remaining two animals. Treadmill running elicited increased phasic recruitment of the PS muscle in all 12 rats studied (Table 3) and was also often associated with some tonic activation. Cessation of locomotion was associated with an abrupt reduction in PS activation in all rats studied.
During the pre-exercise period, the OH and SH muscles were usually silent and exhibited no phasic respiratory recruitment, although large activation of both infrahyoid muscles studied was noted during postural, grooming, or exploratory related movements before exercise. Neither treadmill running, nor stoppage of the treadmill produced any consistent activation of the OH compared to that noted during the nonexercise period. In contrast, treadmill running was associated with increased activation (tonic or phasic expiratory) burst of the SH in five of the six animals studied. In addition, the recruitment of the SH noted during exercise was markedly reduced with stoppage of the treadmill, suggesting that part of the increased recruitment noted in the SH during exercise was due to locomotion. The DG muscle exhibited small tonic activity with occasional bursts that were unrelated to phasic breathing events before exercise. Treadmill running revealed no phasic activation of the DG, but tonic activation was detected in four of six animals studied during exercise. Stoppage of treadmill running was associated with a consistent decrease in DG activation. In comparison, the GG was phasically active, albeit at very low levels, during inspiration during the pre-exercise period in five of the six rats studied. At rest, however, larger EMG bursts were often noted that were related to postural, grooming, or exploratory behavior. Treadmill running produced little change in the phasic recruitment of the GG, whereas the effect on the burst activation was inconsistent across animals studied and appeared little affected by stoppage of the treadmill. All four upper respiratory muscles studied appeared to be activated in response to postural, grooming, feeding, or exploratory behavior to a greater extent than that noted during exercise. Our results are summarized for all four muscles studied in Table 3. Note, for example, that drinking elicited recruitment of the DG and GG muscles that exceeded any recruitment noted during exercise (Figure 2).
Overview of Principal Findings Our data indicate that chronic endurance exercise training is associated with metabolic adaptations in two major upper airway muscles (i.e., DG and SH). Major exercise-induced changes in the DG and SH muscles include (1) an increased oxidative capacity; (2) elevated antioxidant enzyme activity; (3) reduced lipid peroxidation; and (4) a relative increase in Type I MHC content and a relative decrease in Type IIb content. Similar training-induced changes were also observed in the costal diaphragm. In contrast, endurance exercise training did not significantly affect these parameters in the GG or the OH.
Exercise and Oxidative Capacity of Airway Muscles Our hypothesis that the oxidative capacity of these four upper airway muscles would adapt similarly to that of the DIA in response to endurance training was only partially supported by our data. Indeed, compared with control animals, endurance exercise did not alter CS activity in the OH and GG muscles. In contrast, CS activity was elevated in the SH and DG muscles of exercise-trained animals. This observation indicates that regular treadmill exercise is associated with increased recruitment of these muscles either during exercise or perhaps during recovery from exercise. The EMG data collected in these experiments provide qualitative insight as to the nature of the recruitment of these muscles during exercise but does not provide information about the activation pattern of these muscles during nonexercise periods. Our EMG recordings did not detect phasic recruitment of the four upper airway muscles during any phase of the respiratory cycle (Table 3; Figure 2). However, a consistent increase in tonic activity was detected during exercise in both the SH and DG muscles. This type of tonic activity was continuous throughout the exercise bout and suggests that these muscles were used to stabilize the upper airway or neck position during exercise. Hence, it is tempting to speculate that the training stimulus for these upper airway muscles originated from functions that were not entrained with ventilation, but may play a significant respiratory role. However, we cannot rule out the possibility that these upper airway muscles were recruited to perform nonrespiratory tasks during recovery from exercise. For example, possible nonventilatory activities that could be associated with increased contractile activity in upper airway muscles include postural changes, increased eating/drinking behavior, thermoregulation, and even contact with the electrical grid (17) (Table 3). Indeed, the largest activation of these muscles was consistently observed during grooming/thermoregulatory behavior before, or soon after, exercise. Which of these nonventilatory behaviors, if any, contributed to the training adaptation in these airway muscles is difficult to predict from the current data alone. Regardless of the stimulus responsible for the increase in CS activity in the SH and DG muscles, the current data unequivocally demonstrate that exercise training is associated with a training effect in some of the respiratory muscles of the upper airway. Upregulation of CS activity is indicative of a greater oxidative capacity and is correlated to increased fatigue resistance (18). Given that the DG and the SH muscles move the hyoid bone caudally to widen the aperture of the larynx (19), improving the endurance of these muscles should be physiologically advantageous in maintaining airway patency. Note that our failure to observe a consistent and marked increase in EMG activity within the GG muscle of exercising rats differs from recent data on humans. Although some controversy exists, recent work by Williams and colleagues (20) indicate that neural drive to the GG muscle is increased during exercise in humans. The explanation for the apparent species difference in the recruitment of this upper airway is unclear and warrants further study.
Antioxidant Enzyme Activity and Lipid Peroxidation To our knowledge, this is the first study to investigate whether training-induced increases in antioxidant enzyme activities of the upper airway musculature alter the basal levels of lipid peroxidation in resting animals. To evaluate the effects of exercise training on airway muscle lipid peroxidation, we compared the lipid hydroperoxide content of upper airway muscles in both control and trained animals. Our results revealed that the exercise-induced increase in antioxidant enzyme activity (i.e., SOD) was associated with a reduction in lipid peroxidation in the DG and the SH. Although GPX activity was not significantly increased in the DIA and the SH muscles of the trained group, the protection afforded by SOD appears adequate to protect against exercise-induced lipid peroxidation. Alternatively, it is possible that lipid peroxidation was blunted by changes in antioxidant elements that were not measured in the current protocol.
Myosin Phenotype Our finding that training results in a fast-to-slow shift in the MHC profile of the costal diaphragm agrees with previous training studies indicating that regular endurance exercise can promote fast-to-slow shifts in diaphragmatic myosin phenotype. Sugiura and coworkers (23, 24) reported that chronic training reduced the expression of Type IIb fast myosin and increased the expression of Types IIa and IId/x. Recent work in our laboratory has also shown an exercise-induced shift toward increased expression of Type I myosin in the costal diaphragm (4).
Conclusion Although it is clear that endurance exercise training resulted in metabolic adaptations of the SH and DG muscles, the stimulus responsible for this adaptation remains uncertain. EMG recordings from these muscles during exercise indicated an increase in tonic activity but revealed no evidence of phasic recruitment during the respiratory cycle. We also observed that the upper airway muscles are activated during nonventilatory behavior such as postural movements, exploratory behavior, grooming, thermoregulatory behavior, and eating/drinking. Therefore, it is possible that the metabolic adaptations in these respiratory upper airway muscles can be attributed to an exercise-induced increase in both respiratory and nonrespiratory tasks.
Supported by a grant from the American Lung Association-Florida (S. K. P.)
This article has an online data supplement, which is accessible from this issue's table of contents online at www.atsjournals.org Received in original form April 26, 2001; accepted in final form April 9, 2002
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ABSTRACT
INTRODUCTION
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