During the inspiratory phase of breathing, flow resistance through the larynx is kept low through active movement of the vocal cords away from the laryngeal midline (3,8). The posterior cricoarytenoid (PCA) muscle is the primary vocal cord abductor, exhibiting phasic inspiratory and tonic expiratory activity (3,22,27). The onset of phasic PCA activity occurs just before the generation of inspiratory airflow and extends well into the expiratory phase of breathing (22), thereby unloading the diaphragm by reducing the resistance it must work against to inflate the lungs (8).
The glottic dilating action of the PCA muscle rises above resting levels during periods of hyperpnea (22) keeping laryngeal resistance low even when the ventilatory demands imposed on the diaphragm are increased. As a consequence of chemically-evoked hyperpnea, laryngeal resistance decreases to levels below those found during eupnea (4,28). During exercise- induced hyperpnea, mild workloads have been shown to increase the glottic aperture throughout the entire respiratory cycle (9). These wider apertures become more pronounced during severe exertion. Additionally, laryngeal hemiplegia has been shown to increase respiratory muscle work and blood flow during submaximal exercise in the pony (24,25). Although these data reveal that the intrinsic laryngeal muscles play a significant role in minimizing diaphragmatic muscle work during acute hyperpnea, the degree to which they influence the diaphragm in a chronic, physiologically representative situation remains unknown. Determining the influence of the intrinsic laryngeal muscles on the diaphragm would be of value, as loss of vocal cord dilator muscle function does occur in humans (14,21).
The mammalian diaphragm has been shown to be a highly plastic tissue. Studies have shown that metabolic and functional characteristics of the rodent diaphragm are altered following several conditions including undernutrition (20,23,31,33), tracheal banding (19,30,32,35), chronic obstructive lung disease (10,11), and endurance training (15,29). The degree to which the diaphragm’s metabolic profile is influenced by the vocal cord abductors in a chronic, physiologically representative situation remains unknown and formed the basis of our study. The specific purpose of the present study was to determine whether chronic intrinsic laryngeal muscle denervation affected the functional and/or metabolic properties of the diaphragm and whether the additional stimulus of an exercise training protocol brought about additional adaptations. We hypothesized intrinsic laryngeal muscle denervation in sedentary rats would elicit adaptations in the diaphragm that are consistent with increased use, and that these adaptations would be greater in magnitude with the additional stimulus of exercise training.
Thirty-six male Sprague-Dawley rats (initial age, 2 months) were obtained from Harlan Sprague Dawley (Indianapolis, IN) and housed two per cage according to University at Buffalo Laboratory Animal Facility guidelines. Animals were maintained on a 12-h light/dark photoperiod and provided with commercial rat chow and water ad libitum. Upon arrival to the animal facility, the rats were given 7 d to acclimatize to their new surroundings. The experimental protocol was approved by the Institutional Animal Care and Use Committee at SUNY at Buffalo, and accorded with the animal care standards of the American College of Sports Medicine.
Presurgical exercise testing.
After the initial acclimatization period, all animals performed a progressive treadmill exercise test to exhaustion similar to that described by Fregosi and Dempsey (12). The treadmill grade was set at 12° and remained constant throughout the test while running speeds were progressively increased. Initial speed was 15 m·min−1 and increased by 3 m·min−1 every 3 min until the animals could no longer maintain the pace. After completion of the exercise test, animals were randomly assigned either to a control group (sham surgery) or to an intrinsic laryngeal muscle denervation group.
Intrinsic laryngeal muscle denervation.
Denervation of the intrinsic laryngeal muscles was performed according to the method of Maskrey et al. (26). Briefly, each animal was anesthetized with an intraperitoneal injection of a mixture of xylazine (2–10 mg·kg−1) and ketamine (60–80 mg·kg−1). Upon achieving a surgical plane of anesthesia (loss of withdrawal reflex), a vertical incision was made in the midline of the neck, exposing the right and left sternohyoid muscles. Splitting of the sternohyoid permitted identification of the recurrent laryngeal nerves in the groove between the esophagus and the trachea. Small segments (∼2 mm) of the right and left recurrent laryngeal nerves were removed in the denervated animals, whereas the nerves were identified and left intact in the control group. The incision was then closed with silk suture and treated with topical antibiotic. After recovery from the anesthetic, animals were returned to their cages. Intrinsic laryngeal muscle denervation was confirmed by the presence of inspiratory stridor in all denervated, but not control animals.
Postsurgical exercise testing.
After a 2-wk postsurgical recovery period, all animals were again subjected to a progressive treadmill test to exhaustion as described earlier. Animals from the denervated group were then randomly assigned to either a sedentary or an exercise group. It was at this point that we established and will herein refer to our three groups of animals as sedentary control (SC), sedentary denervated (SD), and exercise denervated (ED). Animals from the exercise group began exercise training 3 d after completion of the postsurgical exercise testing.
Endurance exercise training.
ED animals performed a training protocol consisting of treadmill ambulation (0% grade) 6 d·wk−1 for 6 wk. The intensity and duration of the training was progressively increased over the course of the 6-wk exercise period. ED animals were subjected to an exercise regimen that was dictated by the animals’ ability to complete the exercise periods. During the first week of training, animals ambulated for 20 min at 9 m·min−1 with a 3-min warm-up and a 2-min cool-down at 6 m·min−1. Training duration was increased to 30 min during week 2 with no increase in walking speed. Walking speed and duration was 11 m·min−1 and 40 min, respectively, during weeks 3 and 4; 11 m·min−1 and 40 min during week 5; and 12.5 m·min−1 and 50 min during week 6. Warm-up and cool-down periods remained the same throughout the 6 wk of exercise training. On the basis of the mortality and performance of ED animals (see below), training was terminated at 6 wk. After the 6 wk of exercise training, animals from all three groups were again subjected to a third and final progressive treadmill test to exhaustion.
Within 24 h of completing the posttraining exercise test, animals were reanesthetized as described earlier. The soleus muscle was removed in toto, flash frozen in liquid N2, and stored at −70°C. A midline laparotomy was then performed and the costal diaphragm and lower ribs were removed en bloc. The diaphragm and attached ribs were placed in cooled (+4°C) oxygenated Krebs solution containing 137 mM NaCl, 4 mM KCl, 1 mM MgCl2, 1 mM KH2PO4, 12 mM NaHCO3, 2 mM CaCl2, and 6.5 mM glucose and freed from any visible connective tissue. An intact diaphragm bundle with ribs and central tendon attached was dissected free and prepared for measurement of contractile properties. Remaining diaphragm muscle was flash frozen in liquid N2 and stored at −70°C.
Measurement of contractile properties.
In vitro isometric contractile properties of the midcostal diaphragm were measured as previously described (10). Briefly, a single diaphragm bundle was removed, mounted between platinum stimulating electrodes, and placed in a tissue bath containing Krebs solution maintained at 37°C and perfused with 95% O2/5% CO2. After a 15-min thermoequilibration period, optimal length (Lo) was determined with supramaximal monophasic (twitch) pulses of 0.2-ms duration. Muscle length remained fixed at Lo for the measurement of twitch characteristics, force-frequency response, and fatigability.
The muscle bundle was stimulated five times with single supramaximal monophasic pulses of 0.2-ms duration for determination of time to peak tension (TPT), half-relaxation time (1/2 RT), and peak twitch tension (Pt). The muscle bundle was then stimulated for 500 ms at frequencies ranging from 10 to 120 Hz. From these results, a force-frequency curve was plotted and peak tetanic tension (Po) measured. Finally, fatigability was assessed by repetitively stimulating the bundle once per second for 350 ms at a stimulation frequency of 35 Hz for a total duration of 2 min. At the completion of the fatigue test, the bundles were removed from the apparatus, and the diaphragm was cleared of rib and tendinous attachments, blotted dry, and weighed. Cross-sectional area was calculated as muscle weight/(Lo × 1.056) (6).
Maximal citrate synthase (CS) activity was measured in the soleus muscle to identify a peripheral effect consistent with increased use. CS activity was also measured in midcostal diaphragm samples to identify biochemical changes consistent with a training effect. While on ice, muscle samples were cleared of any visible connective tissue and minced with scissors. The minced muscle was then mixed with a homogenizing medium (1:19) and homogenized on ice using a motor-driven glass pestle. The homogenizing medium consisted of 50 mM potassium phosphate (pH 7.4), 1 mM EDTA, 2 mM MgCl2, 2 mM ADP, and 0.5 mM dithiothreitol. CS activity was measured spectrophotometrically at a wavelength of 412 nm at 30°C (34).
All analyses presented in this article include only data from animals completing the entire experimental protocol. Dependent t-tests were used to compare preoperative and postoperative maximal running speeds and body weight gain. Statistical comparisons for all other variables were made by use of a two-way ANOVA, exercise and denervation serving as the independent variables. P ≤ 0.05 was considered significant with Bonferroni post hoc test used to identify specific group differences.
The final number of animals included in the results is given in Table 1. On day 1 of the study (surgery day), of the 36 animals initially acquired, 10 were randomly assigned to the control group and 26 were assigned to the denervated group. By the start of the exercise training (20 d after surgery), 5 of 26 (19%) of the denervated animals had died (four of five deaths occurred within 72 h of the operation) compared with no losses in the control sham-operated animals. On day 20 of the study, the remaining denervated animals were subdivided randomly into sedentary and exercise groups. During the course of the training regimen, none of the sham-operated and none of the sedentary denervated animals died. However, the exercise training, despite its low intensity (see below) appeared to be a formidable stress for the denervated animals, and 5 of 12 animals (42% mortality) died during the course of the training period.
Before the surgery, the denervated animals, despite being randomly assigned, weighed significantly more (P ≤ 0.05) than the control animals (Table 1). After the surgery, however, the animals from both denervated groups weighed less than the controls (Fig. 1). Final body weight for the three groups is also provided in Table 1. During the 9-wk course of the study, animals in the control group increased body weight significantly, whereas animals in both denervated groups showed no significant change in weight (Table 1).
Running speed at exhaustion.
Treadmill speed at exhaustion averaged 27 ± 2 m·min−1 in both groups before the surgery (NS). Fourteen days after surgery, running speed at exhaustion was unchanged in the control animals (25 ± 2 m·min−1), but was significantly reduced (P ≤ 0.05) in the denervated animals (18 ± 3 m·min−1). Running speed at exhaustion for animals of the control and two denervated groups remained unchanged with time or with treadmill training. At the completion of the training protocol, running speeds at exhaustion were 28 ± 2 in the SC group, 19 ± 2 m·min−1 in the SD group, and 22 ± 2 m·min−1 in the ED group.
Diaphragm contractile properties.
Table 2 presents comparisons of Lo, TPT, 1/2 RT, twitch force (Pt), maximal tetanic force (Po), and cross-sectional area (CSA) of the costal diaphragm between the three groups. Intrinsic laryngeal muscle denervation had a significant effect by prolonging TPT and decreasing Po without any additional effects from exercise training. No significant differences were observed in any of the other parameters listed in Table 2.
We present results of the costal diaphragm force-frequency plots in two ways. In absolute terms (Fig. 2A), intrinsic laryngeal muscle denervation had a significant effect on reducing the forces generated at 35, 50, 70, 100, and 120 Hz. There were no differences in the absolute force generated at any other stimulation frequency. When the force-frequency curve is expressed as a percent maximum (Fig. 2B), the relative force generated at 100 Hz was reduced by 1.5% (P ≤ 0.05) in the denervated animals. Exercise training did not affect the diaphragm force-frequency results.
Results from the diaphragm fatigue tests are also presented in two ways in Figure 3. In absolute terms (Fig. 3A), intrinsic laryngeal muscle denervation reduced the force generated by the diaphragm muscle bundles for the first 30 s of the test only. In relative terms (Fig. 3B), diaphragm bundles from denervated animals generated significantly higher forces from 50 s into the test and at all time points thereafter. Exercise training had no independent effect on the fatigue test responses.
Maximal citrate synthase levels in the soleus muscle from the three groups of animals are shown graphically in Figure 4A. Despite the training regimen to which the denervated animals were subjected, exercise training did not produce an up-regulation of citrate synthase activity in the soleus.
A comparison of maximal citrate synthase levels in the costal diaphragm between the three groups is depicted in Figure 4B. Results reveal that denervation of intrinsic laryngeal muscles significantly increased citrate synthase activity in the costal diaphragm without an independent effect of exercise training.
Our data reveal that intrinsic laryngeal muscle denervation caused increases in costal diaphragmatic oxidative capacity and relative in vitro fatigue resistance in sedentary animals, and that 6 wk of treadmill exercise training caused no additional adaptation. Our present findings on mortality, weight gain, and exercise performance demonstrate the importance of glottic dilation during ventilation both at rest and during exercise.
Since the principal laryngeal dilating muscle, the PCA, exhibits both a phasic inspiratory and tonic expiratory activation (3), airway resistance would increase in denervated animals during both phases of the breathing cycle as compared with control conditions. Changes in resistance will affect breathing pattern at rest and during exercise. Maskrey et al. (26) reported that in response to bilateral transection of the recurrent laryngeal nerves, resting rats breathing room air maintain minute ventilation by adopting a breathing pattern with an increased frequency and a reduced tidal volume. These authors reported, however, that denervated rats were able to maintain alveolar ventilation, at least during rest, and concluded that the degree of obstruction caused by paralysis of the intrinsic laryngeal muscles would not appreciably impair gas exchange in the resting state. Maskrey et al. (26) conducted studies 24 h after denervation, whereas the current studies were carried out over a 9-wk period after denervation. Thus, acute and chronic events may be different.
Effects on survival.
Similar to previous reports in tracheal banded rodents (19,30,32,35), we noted that denervation of the intrinsic laryngeal muscles resulted in a high mortality rate. The results reported herein were only completed on survivors. The nature of the high mortality figures is unknown, but may be attributable to respiratory failure. Contrasting the current mortality rate following intrinsic laryngeal muscle denervation with zero mortality seen following bilateral diaphragmatic denervation (17), one is left with the impression that the respiratory system is better designed to function after bilateral loss of the diaphragm than following bilateral loss of the vocal cord abductors.
Effects on body weight.
Denervation of the intrinsic laryngeal muscles had a profound effect on body weight (Fig. 1). During the course of the 9-wk study, the control group increased body weight by approximately 40%, whereas both denervated groups showed either little weight gain or actual weight loss (Fig. 1). Whether the failure to grow in denervated animals is because of decreases in caloric intake and/or increased energy expenditure was not investigated. The losses of body weight reported herein are akin to previous reports of losses of weight in emphysematous hamsters (10), in rats following tracheal banding (19,30,35), and in rats after malnourishment (20,33). In contrast, we previously noted that body weight gain is not affected in rats following bilateral denervation of the diaphragm (17).
Although protein-calorie malnutrition decreases limb muscle oxidative enzymes, the adaptive response of muscle to increased functional demands are unchanged (13). Thus, the main purpose of the study (i.e., to study the plasticity of the diaphragm following denervation of the intrinsic laryngeal muscles) should not have been compromised by the weight loss noted in denervated animals.
Effect on exercise capacity and peripheral training effect.
Denervation of the intrinsic laryngeal muscles significantly reduced running speed at exhaustion. Furthermore, 6 wk of exercise training did not improve running speed at exhaustion. Although we have no direct evidence, we predict that the exercise limitation was respiratory in origin. Airflow limitation, even that of mild to moderate degree, has been shown to limit exercise capacity (2). The reduction in running speed at exhaustion following vocal cord abductor denervation (30%) is similar to that seen after bilateral hemidiaphragm denervation (17) and is consistent with findings that demonstrate a reduction in the ability of exercising ponies to maintain a gallop following laryngeal hemiplegia (24).
Denervated animals, despite being exposed to the 6-wk exercise training protocol, showed no up-regulation of soleus citrate synthase activity that would be consistent with a training effect (Fig. 4A). However, the intent of the exercise was not to induce a peripheral training effect per se, but to increase ventilation. Although the failure to demonstrate a peripheral training effect was probably a result of the low ambulating speed and grade (and therefore only a light stimulus to limb muscle), it seemed to provide a formidable stress to the respiratory muscles. Although no measures of ventilation or work of breathing were obtained during the training sessions themselves, it was visibly evident that respiratory muscle work was quite exaggerated during exercise. Therefore, the lack of a peripheral training effect does not prevent us from addressing the second purpose of the present study, which was to determine whether the additional stimulus of exercise training (hyperpnea) elicits adaptations in the diaphragm above and beyond those seen with ILM denervation.
Effects on diaphragm muscle.
Similar to reports on tracheal banded rodents (30), costal diaphragm bundles following laryngeal muscle denervation exhibited a slower profile compared with controls (Table 2). A prolonged TPT noted in both denervated groups suggests a shift to a greater proportion of slow myosin heavy chain (MHC) isoform (5). The Lo of the costal diaphragm was unchanged following intrinsic laryngeal muscle denervation, suggesting that any increase in airway resistance following intrinsic laryngeal muscle denervation was not associated with chronic hyperinflation. These findings are consistent with reports in tracheal banded rodents (30,35). It is nevertheless possible that dynamic hyperinflation may have occurred during exercise training in the denervated group, as any acute hyperinflation during the exercise sessions would not be expected to modify Lo.
Analysis of the diaphragm force-frequency data shows that intrinsic laryngeal muscle denervation brought about decreases in maximal (Table 2) and submaximal absolute force generation at middle to high (≥35 Hz) stimulation frequencies (Fig. 2A). No decrease in twitch force (Table 2) or in submaximal forces at frequencies lower than 35 Hz were detected (Fig. 2A). These findings would be consistent with selective atrophy of type II fibers, which has been noted following tracheal banding (30). Although controversial, a possible shift in MHC to a slower profile would be consistent with the decreased in vitro diaphragm force generation (16,18). Sarcomeric disruption and/or Z-line streaming, as has been shown to occur in tracheal banded rodents (32), could have contributed to the decreased Po in denervated animals. The dramatic difference in body weight likely played little role, if any, in affecting Po, as malnourishment has been shown to have no effect on maximal tetanic tension when corrected for cross-sectional area (23,31).
Diaphragmatic fatigue resistance (Fig. 3B) was increased 9 wk after intrinsic laryngeal muscle denervation. Similar findings have been reported in emphysematous hamsters (10) and tracheal banded rodents (30,35). The increased in vitro endurance could be attributed to the up-regulation of oxidative enzymes (Fig. 4B) noted in the costal diaphragm, although increases in oxidative enzymes are not a prerequisite for increasing in vitro diaphragmatic endurance to repetitive stimulation (33).
Compared with controls, costal diaphragmatic oxidative capacity increased 20% in denervated animals. This increase in diaphragmatic oxidative capacity is similar to that reported in emphysematous hamsters (11) and endurance-trained rodents (15), but approximately half of increases reported in tracheal banded rodents (19) and sheep intermittently trained with inspiratory resistive loads (1). The intensity of ventilatory loading and/or interspecies differences likely account for these differences in that the ventilatory loads are likely greater following tracheal banding than from intrinsic laryngeal muscle denervation. The decreased body weight that accompanied denervation probably did not influence our results, as malnutrition in rodents has been shown to have no effect on diaphragmatic oxidative capacity (31). Nonetheless, the biochemical data reveal that the intrinsic laryngeal muscles significantly unload the costal diaphragm, even in resting animals, presumably by decreasing the resistance it must work against to inflate the lungs.
Exercise training did not further affect the degree to which the costal diaphragm adapted following intrinsic laryngeal muscle denervation. This finding was unexpected in that the degree to which a limb muscle adapts to a training protocol has been shown to be dependent on the intensity of the stress (7). It would seem likely that the intensity of the ventilatory load in the exercise-denervated animals would be greater than in the sedentary-denervated animals. If the results in limb muscles are carried over to respiratory muscle, one might expect to see higher maximal CS activity levels in the diaphragms of exercise-denervated animals compared with those in the sedentary-denervated group. However, Powers et al. (29) found that training-induced alterations within the costal diaphragm were independent of exercise intensity. These findings may explain why we failed to note a cumulative effect from denervation and exercise training. It is also possible that the additional ventilatory burden of denervation during exercise was, at least in part, met by respiratory muscles other than the costal diaphragm (i.e., crural diaphragm and intercostals), as has been shown to occur during exercise in the laryngeal hemiplegic pony (25). The relatively low speed and intensity of the exercise training protocol, as stated previously, probably accounted for the lack of a training effect in the limb but probably does not explain the lack of a synergistic effect of ILM denervation and exercise training, as the stimulus to ventilation during the bouts, although not measured, was visibly intense.
This study found that 9 wk of intrinsic laryngeal muscle denervation resulted in a more oxidative and a slower diaphragm muscle. The denervation protocol was also associated with reduced diaphragmatic tetanic tension and running speed at exhaustion. An endurance training protocol did not affect the degree to which the rodent costal diaphragm adapted to intrinsic laryngeal muscle denervation. In addition, 42% of the exercise-denervated animals died before data collection. These data highlight the role the intrinsic laryngeal muscles play with regard to unloading the diaphragm by reducing the resistance it must work against to inflate the lungs, especially during exercise. Contrasting the current data with our previous findings following bilateral diaphragm denervation (17), one can infer that the redundancy of the ventilatory pump is such that loss of the main inspiratory muscle, the diaphragm, is better compensated for by other muscles, whereas functional loss of the vocal cord abductor muscles, which has been shown to occur in the human (14,21), has dire consequences on survival, weight gain, and exercise performance.
The authors are grateful to Dr. John A. Krasney for his insights in the preparation of the manuscript.
This work was supported by National Institutes of Health grants HL-43865 and AG-16048.
Address for correspondence: Gaspar A. Farkas, Ph.D., Department of Physical Therapy, Exercise and Nutrition Sciences, 405 Kimball Tower, University of Buffalo, 3435 Main Street, Buffalo, NY 14214-2941; E-mail: firstname.lastname@example.org.
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